Cabozantinib and Tivantinib, but Not INC280, Induce Antiproliferative and Antimigratory Effects in Human Neuroendocrine Tumor Cells in vitro: Evidence for 'Off-Target' Effects Not Mediated by c-Met Inhibition.

BACKGROUND/AIMS: The hepatocyte growth factor/transmembrane tyrosine kinase receptor c-Met has been defined as a potential target in antitumoral treatment of various carcinomas. We aimed to investigate the direct effect of c-Met inhibition on neuroendocrine tumor cells in vitro. METHODS: The effects of the multi-tyrosine kinase inhibitors cabozantinib and tivantinib and of the highly specific c-Met inhibitor INC280 were investigated in human pancreatic neuroendocrine BON1, bronchopulmonary NCI-H727 and midgut GOT1 cells in vitro. RESULTS: INC280, cabozantinib and tivantinib inhibited c-Met phosphorylation, respectively. However, while equimolar concentrations (10 µM) of cabozantinib and tivantinib inhibited cell viability and cell migration, INC280 had no inhibitory effect. Knockdown experiments with c-Met siRNA also did not demonstrate effects on cell viability. Cabozantinib and tivantinib caused a G2 arrest in neuroendocrine tumor cells. CONCLUSIONS: Our in vitro data suggest that c-Met inhibition alone is not sufficient to exert direct antitumoral or antimigratory effects in neuroendocrine tumor cells. The multi-tyrosine kinase inhibitors cabozantinib and tivantinib show promising antitumoral and antimigratory effects in neuroendocrine tumor cells, which are most probably 'off-target' effects, not mediated by c-Met.

Sodium-glucose cotransport inhibitors: mechanisms, metabolic effects and implications for the treatment of diabetic patients with chronic kidney disease.

Remarkable progress has been achieved in the field of diabetes with the development of incretin analogues, dipeptidyl peptidase IV inhibitors and novel insulin analogues; nevertheless, there is an unmet need for additional therapeutic options. Individualization of HbA1c target levels is a recent progress within the field. Approximately 50% of diabetics do not reach a previously aspired treatment goal of glycosylated HbA1 levels below 7% and often face a vicious circle with accelerated weight gain. Current antidiabetic therapeutics mainly target the decline in insulin secretion and ameliorate insulin resistance. In this regard a new generation of drugs, denoted gliflozines, that specifically interfere with sodium-glucose cotransporters (SGLT)-2 and exhibit a favourable impact on glucose metabolism in patients with type 2 diabetes are emerging as hopeful avenues. The resultant negative energy balance caused by glucosuria results in long-term weight losses, significantly reduced HbA1c levels approximating 0.5-1.0% and may in addition exert beneficial effects on blood pressure, reactive oxygen products and inflammatory mediators. Recent studies indicate improvement in ß-cell glucose sensitivity and insulin sensitivity in patients treated with gliflozines, a decrease in tissue glucose disposal and interestingly an increase in endogenous glucose production. The list of side effects observed under SGLT2 inhibition includes increased rates of genitourinary infections, balanitis, vulvovaginitis, hypotensive episodes and acute deterioration of kidney function. Main questions towards the safety profile are still unanswered given that long-term clinical outcome data with SGLT2 inhibition are lacking and the cardiovascular safety profile is under scrutiny in large trials. Thus, the successful development of selective SGLT2 inhibitors for therapeutic use in diabetics has a huge potential to meet patients' needs. However, it awaits quick results from clinical trials with meaningful clinical endpoints.

The novel somatostatin receptor 2/dopamine type 2 receptor chimeric compound BIM-23A758 decreases the viability of human GOT1 midgut carcinoid cells.

The majority of neuroendocrine tumors (NETs) of the gastroenteropancreatic system coexpress somatostatin receptors (SSTRs) and dopamine type 2 receptors (D2R), thus providing a rationale for the use of novel SSTR2/D2R chimeric compounds in NET disease. Here we investigate the antitumor potential of the SSTR2/D2R chimeric compounds BIM-23A760 and BIM-23A758 in comparison to the selective SSTR2 agonist BIM-23023 and the selective D2R agonist BIM-53097 on human NET cell lines of heterogeneous origin. While having only minor effects on human pancreatic and bronchus carcinoid cells (BON1 and NCI-H727), BIM-23A758 induced significant antitumor effects in human midgut carcinoid cells (GOT1). These effects involved apoptosis induction as well as inhibition of mitogen-activated protein kinase and Akt signaling. Consistent with their antitumor response to BIM-23A758, GOT1 cells showed relatively high expression levels of SSTR2 and D2R mRNA. In particular, GOT1 cells highly express the short transcript variant of D2R. In contrast to BIM-23A758, the SSTR2/D2R chimeric compound BIM-23A760 as well as the individual SSTR2 and D2R agonistic compounds BIM-23023 and BIM-53097 induced no or only minor antitumor responses in the examined NET cell lines. Taken together, our findings suggest that the novel SSTR2/D2R chimeric compound BIM-23A758 might be a promising substance for the treatment of NETs highly expressing SSTR2 and D2R. In particular, a sufficient expression of the short transcript variant of DR2 might play a pivotal role for effective treatment.

Pituitary tumor-transforming gene: physiology and implications for tumorigenesis.

Pituitary tumor-transforming gene-1 (PTTG1) is overexpressed in a variety of endocrine-related tumors, especially pituitary, thyroid, breast, ovarian, and uterine tumors, as well as nonendocrine-related cancers involving the central nervous, pulmonary, and gastrointestinal systems. Forced PTTG1 expression induces cell transformation in vitro and tumor formation in nude mice. In some tumors, high PTTG1 levels correlate with invasiveness, and PTTG1 has been identified as a key signature gene associated with tumor metastasis. Increasing evidence supports a multifunctional role of PTTG1 in cell physiology and tumorigenesis. Physiological PTTG1 properties include securin activity, DNA damage/repair regulation and involvement in organ development and metabolism. Tumorigenic mechanisms for PTTG1 action involve cell transformation and aneuploidy, apoptosis, and tumorigenic microenvironment feedback. This paper reviews recent advances in our understanding of PTTG1 structure and regulation and addresses known mechanisms of PTTG1 action. Recent knowledge gained from PTTG1-null mouse models and transgenic animals and their potential application to subcellular therapeutic targeting PTTG1 are discussed.

Anti-aging therapy with human growth hormone associated with metastatic colon cancer in a patient with Crohn's colitis.

BACKGROUND & AIMS: The nonapproved use of human growth hormone (HGH) for anti-aging has been increasing. Theoretical concerns for neoplastic potentiation by HGH have been raised, but not proven clinically. METHODS: We report the case of a 68-year-old man with colonic Crohn's disease who was found to have aggressive metastatic colon cancer. The patient had been receiving HGH therapy for anti-aging purposes for 7 years before presentation. Normal and malignant colonic tissue was examined for qualitative and quantitative molecular profiles of growth hormone (GH) and its signaling molecules, using immunohistochemistry and RNA extraction with polymerase chain reaction amplification. RESULTS: Immunoreactivity was more robust in tumor tissue than in normal colon for insulin-like growth factor-1 receptor (IGF-1R) but not for IGF, GH, or GH receptor. RNA extraction with quantitative polymerase chain reaction showed that IGF-1R and vascular endothelial growth factor expression, but not IGF-1, GH receptor, or suppressor of cytokine signaling-2, were higher in tumor than in normal colonic tissue. CONCLUSIONS: Colorectal cancer development concurrent with administration of HGH for anti-aging purposes occurred in an individual already at increased risk for colon cancer. This underscores the need for further investigation of the proneoplastic potential of GH supplementation for anti-aging.

Novel neurotrophin-1/B cell-stimulating factor-3 (NNT-1/BSF-3)/cardiotrophin-like cytokine (CLC)--a novel gp130 cytokine with pleiotropic functions.

Novel neurotrophin-1/B-cell stimulating factor-3 (NNT-1/BSF-3) is a new member of the gp130 cytokine family. NNT-1/BSF-3 is a second ligand to the tripartite CNTFR complex and activates Jak-STAT, MAPK and PI3/Akt signaling pathways in various cell systems. So far, the known functions of NNT-1/BSF-3 encompass neurotrophic and B cell stimulatory effects, as well as neuroimmunoendocrine modulation of corticotroph function. Gene expression of NNT-1/BSF-3 is stimulated by PKA- and PKC-dependent pathways. Cellular secretion of NNT-1/BSF-3 requires heteromeric complex formation with other factors, e.g. cytokine-like factor-1 (CLF-1) or soluble ciliary neurotrophic factor receptor (sCNTFR). This article reviews the current knowledge on NNT-1/BSF-3 expression, secretion, receptor interaction, signal transduction and physiologic effects of this novel gp130 cytokine. Remark: After preparation of this manuscript, another novel gp130 cytokine named neuropoietin (NP) has been reported and shown to be a ligand of the CNTFR complex.

Modulation of monocytic lipopolysaccharide-induced tissue factor expression and tumor necrosis factor alpha release by estrogen and calcitriol.

OBJECTIVE: Modulatory effects of estrogens on both the immune and the coagulation system are only partially understood. In severe infections high estrogen levels have been observed both in men and postmenopausal women and are associated with increased mortality. Monocyte-derived tissue factor (TF) expression can activate the coagulation system and worsen the course of severe infection. T he aim of the current study was to evaluate the in vitro effect of estrogens on differentiation, TF expression and Tumor Necrosis Factor alpha (TNFalpha) release in human monocytes. DESIGN: Isolated peripheral blood monocytes, MM6- and T HP-1 cells were cultured and stimulated by lipopolysaccharides (LPS) in the presence of 17beta-estradiol (E2) and/or calcitriol. Proliferative responses were evaluated by determining the proliferation rate and by cell cycle analysis. Cell surface expression of C D14 and T F was determined by flow cytometry. TNFalpha was determined by ELISA. RESULTS: Although calcitriol induced the expression of the differentiation marker C D14 and decreased the expression of T F in both immature monocytic cell lines and primary monocytes, the LPS stimulation of T F expression was not significantly increased in immature monocytic cells and was decreased in mature monocytes. Calcitriol-treatment increased LPS-induced TNFalpha release in MM6 cells but inhibited TNFalpha release from peripheral blood monocytes. Treatment with E2 did not alter the phenotype or cell proliferation of resting monocytic cells. However, E2-treated monocytic cells and monocytes responded to LPS by increased TF expression and decreased TNFalpha. CONCLUSIONS: The results suggest that estrogens may modulate T F expression and cytokine production by monocytes and may thus be involved, at least in part, in the pathophysiology of acute inflammatory processes associated with high estrogen levels.

Role of the intracellular receptor domain of gp130 (exon 17) in human inflammatory bowel disease.

AIM: To study the role of the intracellular receptor domain of gp130 in human inflammatory bowel disease (IBD). METHODS: We amplified and sequenced the complete exon 17 of the human gp130 gene in 146 patients with IBD. According to clinical and histopathological signs, the 146 patients with IBD were classified as having Crohn's disease (n = 73) or ulcerative colitis (n = 63), or as indeterminate status (n = 10). RESULTS: No mutations in exon 17 of the gp130 gene could be detected in any of the 146 patients with IBD examined. CONCLUSION: There is no evidence that mutations in exon 17 of the gp130 gene are involved in the pathogenesis of human IBD.

Expression of novel neurotrophin-1/B-cell stimulating factor-3 (NNT-1/BSF-3) in murine pituitary folliculostellate TtT/GF cells: pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide-induced stimulation of NNT-1/BSF-3 is mediated by protein kinase A, protein kinase C, and extracellular-signal-regulated kinase1/2 pathways.

Novel neurotrophin-1/B cell stimulating factor-3 (NNT-1/BSF-3) is a gp130 cytokine potently stimulating corticotroph proopiomelanocortin gene expression and ACTH secretion by a Janus kinase-signal transducer and activator of transcription (JAK-STAT)-dependent mechanism. In the current study, we examined the regulation of NNT-1/BSF-3 mRNA expression in murine pituitary folliculostellate TtT/GF cells using Northern blot technique. A 5- to 9-fold and a 4- to 7-fold induction in NNT-1/BSF-3 mRNA expression was observed between 2 and 6 h stimulation with the protein kinase C (PKC) stimulus phorbol-12-myristate-13-acetate (100 nm) and the protein kinase A (PKA) stimulus Bu(2)cAMP (5 mm), respectively. Pituitary adenylate cyclase-activating polypeptide (PACAP-38, 50 nm) and vasoactive intestinal peptide (VIP, 50 nm) also stimulated NNT-1/BSF-3 mRNA expression 5- to 9-fold between 2 and 6 h. Preincubation with PKC and PKA inhibitors such as H-7 (20 microm), GF109203X (50 microm), and H-89 (50 microm) decreased the stimulatory effects of PACAP and VIP. Both PACAP-38 and VIP also rapidly induced ERK1/2 phosphorylation and their stimulatory effect on NNT-1/BSF-3 mRNA expression was reduced by the MAPK kinase/ERK kinase (MEK) inhibitor U0126 (10 microm). Dexamethasone (10(-7) m) was a potent inhibitor of phorbol-12-myristate-13-acetate-induced NNT-1/BSF-3 expression. RT-PCR analysis demonstrated TtT/GF cells to express the short and the hop variant but not the hip variant of the PACAP-1 receptor (PAC1-R). In addition, TtT/GF cells express the VIP/PACAP-2 receptor (VPAC2-R). In summary, NNT-1/BSF-3 is expressed in pituitary folliculostellate TtT/GF cells and induced by PKC-, PKA-, and ERK1/2-dependent mechanisms. The novel gp130 cytokine NNT-1/BSF-3 derived from folliculostellate cells might act as a paracrine neuroimmunoendocrine modulator of pituitary corticotroph function.

The oncostatin M receptor/gp130 ligand murine oncostatin M induces apoptosis in adrenocortical Y-1 tumor cells.

The effects of murine oncostatin M (mOSM) are specifically mediated by the heterodimeric oncostatin M receptor (OSMR)/gp130 receptor complex. In the current study we demonstrate that murine adrenocortical Y-1 tumor cells express the OSMR/gp130 complex. Incubation of Y-1 cells with 1 and 10 ng/ml mOSM induces cell death due to specific induction of apoptosis. Western blot analysis of Y-1 cells incubated with mOSM for 24 h revealed caspase-3 cleavage and poly(ADP-ribase) polymerase (PARP) cleavage. In a proliferation assay system, incubation of Y-1 cells with 0.01, 0.1, 1 and 10 ng/ml mOSM for 24 h resulted in a decrease in cell numbers to 99+/-2%, 84+/-9%, 50+/-7% and 43+/-5% respectively of untreated control (defined as 100%). Pretreatment of Y-1 cells with the Jak2 inhibitor AG490 (100 microM) rescued Y-1 cells from OSM-induced (10 ng/ml) cell death. Similarly, pretreatment of Y-1 cells with the general caspase inhibitor Z-VAD-FMK (42 microM) rescued Y-1 cells from OSM-induced (10 ng/ml) cell death. In summary, we show that adrenocortical Y-1 tumor cells express the OSMR/gp130 complex and that mOSM induces the Jak-STAT signaling cascade in these cells. Murine OSM in a dose-dependent manner induces apoptosis in adrenocortical Y-1 tumor cells. Apoptosis was demonstrated by caspase-3 cleavage and PARP cleavage. Rescue of Y-1 cells from mOSM-induced apoptosis by the Jak2 inhibitor, AG490, and the general caspase inhibitor, Z-VAD-FMK, demonstrates Jak activation and subsequent caspase activation to be essential for mOSM-induced apoptosis in adrenocortical Y-1 tumor cells. The putative role of OSM as an immunotherapeutic agent in human adrenocortical cancer remains to be elucidated.

Aspirin inhibits cell viability and mTOR downstream signaling in gastroenteropancreatic and bronchopulmonary neuroendocrine tumor cells.

AIM: To investigate the effect of aspirin on neuroendocrine tumor (NET) cell growth and signaling in vitro. METHODS: Human pancreatic BON1, bronchopulmonary NCI-H727 and midgut GOT1 neuroendocrine tumor cells were treated with different concentrations of aspirin (from 0.001 to 5 mmol/L), and the resulting effects on metabolic activity/cell proliferation were measured using cell proliferation assays and SYBR-DNA-labeling after 72, 144 and 216 h of incubation. The effects of aspirin on the expression and phosphorylation of several critical proteins that are involved in the most common intracellular growth factor signaling pathways (especially Akt protein kinase B) and mammalian target of rapamycin (mTOR) were determined by Western blot analyses. Propidium iodide staining and flow cytometry were used to evaluate changes in cell cycle distribution and apoptosis. Statistical analysis was performed using a 2-tailed Student's t-test to evaluate the proliferation assays and cell cycle analyses. The results are expressed as the mean ± SD of 3 or 4 independently performed experiments. Statistical significance was set at P < 0.05. RESULTS: Treatment with aspirin suppressed the viability/proliferation of BON1, NCI-H727 and GOT1 cells in a time- and dose-dependent manner. Significant effects were observed at starting doses of 0.5-1 mmol/L and peaked at 5 mmol/L. For instance, after treatment with 1 mmol/L aspirin for 144 h, the viability of pancreatic BON1 cells decreased to 66% ± 13% (P < 0.05), the viability of bronchopulmonary NCI-H727 cells decreased to 53% ± 8% (P < 0.01) and the viability of midgut GOT1 cells decreased to 89% ± 6% (P < 0.01). These effects were associated with a decreased entry into the S phase, the induction of the cyclin-dependent kinase inhibitor p21 and reduced expression of cyclin-dependent kinase 4 and cyclin D3. Aspirin suppressed mTOR downstream signaling, evidenced by the reduced phosphorylation of the mTOR substrates 4E binding protein 1, serine/threonine kinase P70S6K and S6 ribosomal protein and inhibited glycogen synthase kinase 3 activity. We observed the (compensatory) activation of tuberous sclerosis 2, the serine/threonine specific protein kinase AKT and extracellular signal-regulated kinases. CONCLUSION: Aspirin demonstrates promising anticancer properties for NETs in vitro. Further preclinical and clinical studies are needed.

Anticancer effects of metformin on neuroendocrine tumor cells in vitro.

Metformin is a widely used oral antidiabetic drug with good tolerability. Recent studies suggest that it also possesses adjuvant potent anticancer properties in a variety of tumors. Neuroendocrine tumors (NETs) of the gastro-entero-pancreatic system (GEP) comprise a heterogeneous group of tumors with increasing incidence and limited effective therapeutic options. Here we report the antiproliferative effects of metformin in neuroendocrine tumor cells in vitro. Treatment of human pancreatic BON1, bronchopulmonary NCI-H727, and midgut GOT1 neuroendocrine tumor cells with increasing concentrations of metformin (0.1-10 mM) dose-dependently suppressed cell viability and cell counts. Metformin induced AMPK phosphorylation in pancreatic BON1 and midgut GOT1 but suppressed AMPK activity in bronchopulmonary NCI-H727. Thus, AMPK-dependent and AMPK-independent properties may be operative in NETs of different origin. Metformin suppressed mTORC1 signaling in all three tumor cell types, evidenced by suppression of 4EBP1, pP70S6K, and S6 phosphorylation, and was associated with compensatory AKT activity. We observed induction of ERK phosphorylation in BON1 and NCI-H727 and inhibition of ERK in midgut GOT1 cells, while all three tumor cell types responded with induction of GSK3 phosphorylation. This suggests a central role for GSK3 in metformin-mediated signal transduction. Inhibition of cell proliferation by metformin was associated with apoptosis induction only in midgut GOT1, evidenced by increased subG0/1 fraction and PARP cleavage. These results suggest a potential role of metformin as a (adjuvant) therapeutic for patients with NETs.

Combination of 13-Cis retinoic acid and lovastatin: marked antitumor potential in vivo in a pheochromocytoma allograft model in female athymic nude mice.

Currently, there are no reliably effective therapeutic options for metastatic pheochromocytoma (PCC) and paraganglioma. Moreover, there are no therapies that may prevent the onset or progression of tumors in patients with succinate dehydrogenase type B mutations, which are associated with very aggressive tumors. Therefore, we tested the approved and well-tolerated drugs lovastatin and 13-cis-retinoic acid (13cRA) in vitro in an aggressive PCC mouse cell line, mouse tumor tissue-derived (MTT) cells, and in vivo in a PCC allograft nude mouse model, in therapeutically relevant doses. Treatment was started 24 hours before sc tumor cell injection and continued for 30 more days. Tumor sizes were measured from outside by caliper and sizes of viable tumor mass by bioluminescence imaging. Lovastatin showed antiproliferative effects in vitro and led to significantly smaller tumor sizes in vivo compared with vehicle treatment. 13cRA promoted tumor cell growth in vitro and led to significantly larger viable tumor mass and significantly faster increase of viable tumor mass in vivo over time compared with vehicle, lovastatin, and combination treatment. However, when combined with lovastatin, 13cRA enhanced the antiproliferative effect of lovastatin in vivo. The combination-treated mice showed slowest tumor growth of all groups with significantly slower tumor growth compared with the vehicle-treated mice and significantly smaller tumor sizes. Moreover, the combination-treated group displayed the smallest size of viable tumor mass and the slowest increase in viable tumor mass over time of all groups, with a significant difference compared with the vehicle- and 13cRA-treated group. The combination-treated tumors showed highest extent of necrosis, lowest median microvessel density and highest expression of α-smooth muscle actin. The combination of high microvessel density and low α-smooth muscle actin is a predictor of poor prognosis in other tumor entities. Therefore, this drug combination may be a well-tolerated novel therapeutic or preventive option for malignant PCC.

Potent antitumor activity of the novel HSP90 inhibitors AUY922 and HSP990 in neuroendocrine carcinoid cells.

The heat shock protein (HSP) 90 chaperone machine involved in numerous oncogenic signaling pathways is overexpressed in cancer cells and is currently being evaluated for anticancer therapy. Neuroendocrine tumors (NETs) of the gastroenteropancreatic (GEP) system comprise a heterogeneous group of tumors with increasing incidence and poor prognosis. Here, we report the antiproliferative effects of the HSP90 inhibitors AUY922 and HSP990 in neuroendocrine tumor cells. Treatment of human pancreatic BON1, bronchopulmonary NCI-H727 and midgut carcinoid GOT1 neuroendocrine tumor cells with increasing concentrations of AUY922 and HSP990 dose-dependently suppressed cell viability. Significant effects on neuroendocrine cell viability were observed with inhibitor concentrations as low as 5 nM. Inhibition of cell viability was associated with the induction of apoptosis as demonstrated by an increase in sub-G1 events and PARP cleavage. HSP90 inhibition was associated with decreased neuroendocrine ErbB and IGF-I receptor expression, decreased Erk and Akt phospho-rylation and the induction of HSP70 expression. These findings provide evidence that targeted inhibition of upregulated HSP90 activity could be useful for the treatment of aggressive neuroendocrine tumors resistant to conventional therapy.

Hypoxia-induced VEGF production 'RSUMEs' in pituitary adenomas.

Angiogenic markers in pituitary adenomas remain enigmatic in terms of their function in tumorigenesis, despite being upregulated by the normal physiological trigger of hypoxia. In this issue of Endocrine-Related Cancer, Shan et al. report that the novel RWD domain containing protein, RWD-containing sumoylation enhancer, is expressed in human pituitary adenomas and plays a pivotal role in regulating the hypoxia-inducible factor 1α-vascular endothelial growth factor response to hypoxia.

Perifosine-mediated Akt inhibition in neuroendocrine tumor cells: role of specific Akt isoforms.

The majority of neuroendocrine tumors (NETs) of the gastroenteropancreatic system show aberrant Akt activity. Several inhibitors of the phosphoinositide 3-kinase (PI(3)K)-Akt-mTOR signaling pathway are currently being evaluated in clinical phase II and III studies for the treatment of NETs with promising results. However, the molecular mechanisms and particularly the role of different Akt isoforms in NET signaling are not fully understood. In this study, we examine the effect of Akt inhibition on NET cells of heterogeneous origin. We show that the Akt inhibitor perifosine effectively inhibits Akt phosphorylation and cell viability in human pancreatic (BON1), bronchus (NCI-H727), and midgut (GOT1) NET cells. Perifosine treatment suppressed the phosphorylation of Akt downstream targets such as GSK3α/ß, MDM2, and p70S6K and induced apoptosis. To further investigate the role of individual Akt isoforms for NET cell function, we specifically blocked Akt1, Akt2, and Akt3 via siRNA transfection. In contrast to Akt2 knockdown, knockdown of Akt isoforms 1 and 3 decreased phosphorylation levels of GSK3α/ß, MDM2, and p70S6K and suppressed NET cell viability and colony-forming capacity. The inhibitory effect of simultaneous downregulation of Akt1 and Akt3 on tumor cell viability was significantly stronger than that caused by downregulation of all Akt isoforms, suggesting a particular role for Akt1 and Akt3 in NET signaling. Akt3 siRNA-induced apoptosis while all three isoform-specific siRNAs impaired BON1 cell invasion. Together, our data demonstrate potent antitumor effects of the pan-Akt inhibitor perifosine on NET cells in vitro and suggest that selective targeting of Akt1 and/or Akt3 might improve the therapeutic potential of Akt inhibition in NET disease.

Expression and function of ErbB receptors and ligands in the pituitary.

The role of ErbB family in discreet pituitary functions is reviewed. Several ErbB receptor ligands, EGF, TGFα, and heregulin are differentially expressed in normal gonadotroph and lacto-somatotroph lineages, and other elements of the anterior pituitary. ErbB receptors, i.e. EGFR and ErbB2, are also localized to the anterior pituitary with preferential EGFR lactosomatotroph expression. EGF regulates CRH and ACTH secretion and corticotroph proliferation as well as exhibiting autocrine and paracrine effects on gonadotrophs and on lactosomatotroph proliferation, gene and protein expression, and hormonal secretion. EGF and EGFR are expressed in both functioning and non-functioning pituitary adenomas, with higher expression in more aggressive tumor subtypes. ErbB2 receptor is detected in all tumor subtypes, particularly in invasive tumors. ErbB tyrosine kinase inhibitors regulate hormonal secretion, cell morphology, and proliferation in lacto-somatotroph tumors, reflecting the emerging application of targeted pituitary therapeutics.

Fibroblast growth factor-2 autofeedback regulation in pituitary folliculostellate TtT/GF cells.

To investigate paracrine regulation of pituitary cell growth, we tested fibroblast growth factor (FGF) regulation of TtT/GF folliculostellate (FS) cells. FGF-2, and FGF-4 markedly induced cell proliferation, evidenced by induction of pituitary tumor transforming gene-1 (Pttg1) mRNA expression and percentage of cells in S phase. Signaling for FGF-2-induced FS cell proliferation was explored by specific pharmacological inhibition. A potent inhibitory effect on FGF-2 action was observed by blocking of Src tyrosine kinase with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (>or=0.1 microm), followed by protein kinase C (PKC) inhibition with GF109203X. Treatment with FGF-2 (30 ng/ml; 10 min) activated phosphorylation of signal transducer and activator of transcription-3, ERK, stress-activated protein kinase/c-Jun N-terminal kinase, Akt, and focal adhesion kinase. Src inhibition with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine suppressed FGF-2-induced Akt and focal adhesion kinase, indicating effects downstream of FGF-2-induced Src activation. FGF-2 also markedly induced its own mRNA expression, peaking at 2-4 h, and this effect was suppressed by Src tyrosine kinase inhibition. The PKC inhibitor GF109203X abolished FGF-2 autoinduction, indicating PKC as the primary pathway involved in FGF-2 autoregulation in these cells. In addition to pituitary FGF-2 paracrine activity on hormonally active cells, these results show an autofeedback mechanism for FGF-2 in non-hormone-secreting pituitary FS cells, inducing cell growth and its own gene expression, and mediated by Src/PKC signaling.

Heregulin regulates prolactinoma gene expression.

To investigate the role of p185(her2/neu)/ErbB3 signaling in pituitary tumor function, we examined these receptors in human prolactinomas. Immunofluorescent p185(her2/neu) was detected in almost all (seven of eight), and ErbB3 expression in a subset (four of eight) of tumors (seven adenomas and one carcinoma). Quantitative PCR also showed abundant ErbB3 mRNA in tumor specimens derived from a rarely encountered prolactin-cell carcinoma. Activation of p185(c-neu)/ErbB3 signaling with heregulin, the ErbB3 ligand, in rat lacto-somatotroph (GH4C1) tumor cells specifically induced prolactin (PRL) mRNA expression approximately 5-fold and PRL secretion approximately 4-fold, whereas growth hormone expression was unchanged. Heregulin (6 nmol/L) induced tyrosine phosphorylation and ErbB3 and p185(c-neu) heterodimerization, with subsequent activation of intracellular ERK and Akt. The Akt signal was specific to ErbB3 activation by heregulin, and was not observed in response to epidermal growth factor activation of epidermal growth factor receptor. Gefitinib, the tyrosine kinase inhibitor, suppressed heregulin-mediated p185(c-neu)/ErbB3 signaling to PRL. Heregulin induction of PRL was also abrogated by transfecting cells with short interfering RNA directed against ErbB3. Pharmacologic inhibition of heregulin-induced phosphoinositide-3-kinase/Akt (with LY294002) and ERK (with U0126) signaling, as well as short interfering RNA-mediated mitogen-activated protein kinase-1 down-regulation, showed ERK signaling as the primary transducer of heregulin signaling to PRL. These results show ErbB3 expression in human prolactinomas and a novel ErbB3-mediated mechanism for PRL regulation in experimental lactotroph tumors. Targeted inhibition of up-regulated p185(c-neu)/ErbB3 activity could be useful for the treatment of aggressive prolactinomas resistant to conventional therapy.

PI(3)K-Akt-mTOR pathway as a potential therapeutic target in neuroendocrine tumors.

Constitutive activation of PI(3)K-Akt-mTOR signaling is a frequently occurring event in human cancer and has also been detected in the majority of neuroendocrine tumors (NETs) of the gastroenteropancreatic system. Molecular analysis of NETs suggests, that in addition to mutations in certain tumor-suppressor genes (e.g., PTEN), multiple autocrine growth factor loops contribute to hyperactive PI(3)K-Akt-mTOR signaling, thus promoting unrestricted proliferation and resistance to apoptosis. These insights opened new perspectives for targeted therapy in NETs. In particular, several novel small-molecule inhibitors of tyrosine and serine/threonine kinases have demonstrated potent anti-tumor activity. This review will summarize current knowledge on PI(3)K-Akt-mTOR signaling, its role in proliferation and apoptosis, as well as novel therapeutic approaches targeting PI(3)K-Akt-mTOR pathway components in NET disease.