The Novel Cyclin-Dependent Kinase 4/6 Inhibitor Ribociclib (LEE011) Alone and in Dual-Targeting Approaches Demonstrates Antitumoral Efficacy in Neuroendocrine Tumors in vitro

BACKGROUND/AIM Cyclin-dependent kinases (CDKs) are crucial for cell cycle regulation, and alterations in the cell cycle are often observed in human cancer. CDK4/6 in particular orchestrates G1 phase progression and the G1/S transition. Here, we investigated the in vitro effects of the CDK4/6 inhibitor LEE011 in human neuroendocrine tumor cells. METHODS The human neuroendocrine tumor cell lines BON1, QGP1, NCI-H727 and GOT1 were treated with different concentrations of LEE011 alone and in combination with 5-fluorouracil and everolimus. RESULTS Cell viability decreased in a time- and dose-dependent manner in BON1, QGP1, and NCI-H727 cells upon LEE011 treatment, whereas GOT1 cells were treatment resistant. Treatment sensitivity towards LEE011 was associated with the high expression of cyclin D1 and Rb. LEE011 caused the dephosphorylation of Rb and a subsequent G1 phase cell cycle arrest. Combined treatment with LEE011 and 5-fluorouracil or everolimus showed a significant enhancement in the inhibition of cell viability when compared to single-substance treatments due to PI3K-Akt-mTOR and Ras-Raf-MEK-ERK pathway downregulation and cooperative downregulation of cell cycle components. However, LEE011 also exhibited antagonizing effects with 5-fluorouracil, protecting NET cells from DNA-damaging chemotherapy by blocking PARP cleavage and caspase-3/7 activity. CONCLUSIONS Our data demonstrate that the CDK 4/6 inhibitor LEE011 exhibits promising anti-tumoral properties alone and in combination treatment approaches with 5-fluorouracil or everolimus in human neuroendocrine tumor cell lines

The Role of the Octarepeat Region in Neuroprotective Function of the Cellular Prion Protein

Structural alterations of the cellular prion protein (PrP(C)) seem to be the core of the pathogenesis of prion diseases. However, the physiological function of PrP(C) remains an enigma. Cell culture experiments have indicated that PrP(C) and in particular its N-terminal octarepeat region together with the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways have a fundamental involvement in neuroprotection and oxidative stress reactions. We used wild-type mice, PrP knockout (Prnp(−/−)) animals and transgenic mice that lack the octarepeat region (C4/−) and subjected them to controlled ischemia. We identified an increased cleavage and synthesis of PrP(C) in ischemic brain areas of wild-type mice compared with sham controls. The infarct size in Prnp(−/−) animals was increased threefold when compared with wild-type mice. The infarct size in C4/− animals was identical to Prnp(−/−) mice, that is, around three times larger than in wild-type mice. We showed that the PrP in C4/− mice does not functionally rescue the Prnp(−/−) phenotype; furthermore it is unable to undergo β cleavage, although an increased amount of C1 fragments was found in ischemic brain areas compared with sham controls. We demonstrated that the N-terminal octarepeat region has a lead function in PrP(C) physiology and neuroprotection against oxidative stress in vivo

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 mu M) 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. (C) 2015 S. Karger AG, Base

The MTH1 inhibitor TH588 demonstrates anti-tumoral effects alone and in combination with everolimus, 5-FU and gamma-irradiation in neuroendocrine tumor cells

Modulation of the redox system in cancer cells has been considered a promising target for anti-cancer therapy. The novel MTH1 inhibitor TH588 proved tremendous potential in terms of cancer cell eradication, yet its specificity has been questioned by recent reports, indicating that TH588 may also induce cancer cell death by alternative mechanisms than MTH1 inhibition. Here we used a panel of heterogeneous neuroendocrine tumor cells in order to assess cellular mechanisms and molecular signaling pathways implicated in the effects of TH588 alone as well as dual-targeting approaches combining TH588 with everolimus, cytotoxic 5-fluorouracil or Y-irradiation. Our results reflect that TH588 alone efficiently decreased the survival of neuroendocrine cancer cells by PI3K-Akt-mTOR axis downregulation, increased apoptosis and oxidative stress. However, in the dual-targeting approaches cell survival was further decreased due to an even stronger downregulation of the PI3K-Akt-mTOR axis and augmentation of apoptosis but not oxidative stress. Furthermore, we could attribute TH588 chemo- and radio-sensitizing properties. Collectively our data not only provide insights into how TH588 exactly kills cancer cells but also depict novel perspectives for combinatorial treatment approaches encompassing TH588

Immuno-histochemical correlation of fibrosis-related markers with the desmoplastic reaction of the mesentery in small intestine neuroendocrine neoplasms

INTRODUCTION Small intestine neuroendocrine neoplasms (siNENs) will attain more importance due to their increasing incidence. Moreover, siNENs might lead to a desmoplastic reaction (DR) of the mesentery causing severe complications and deteriorating prognosis. The expression of fibrosis-related proteins appears to be the key mechanisms for the development of this desmoplastic reaction. Therefore, this study aimed to investigate the association of the desmoplastic mesentery with specific fibrosis-related protein expression levels. MATERIALS AND METHODS By immunohistochemistry, the protein expression levels of four fibrosis-related markers (APLP2, BNIP3L, CD59, DKK3) were investigated in primary tumors of 128 siNENs. The expression levels were correlated with the presence of a desmoplastic reaction and clinico-pathological parameters. RESULTS In the primary tumor, APLP2, BNIP3L, CD59 and DKK3 were highly expressed in 29.7% (n = 38), 64.9% (n = 83), 92.2% (n = 118) and 80.5% (n = 103), respectively. There was no significant correlation of a single marker or the complete marker panel to the manifestation of a desmoplastic mesentery. The desmoplastic mesentery was significantly associated with clinical symptoms, such as flushing and diarrhea. However, neither the fibrosis-related marker panel nor single marker expressions were associated with clinical symptoms. DISCUSSION The expression rates of four fibrosis-related markers in the primary tumor display a distinct pattern. However, the expression patterns are not associated with desmoplastic altered mesenteric lymph node metastases and the expression patterns did not correlate with prognosis. These findings suggest alternative mechanisms being responsible for the desmoplastic reaction

Innovative multidimensional models in a high-throughput-format for different cell types of endocrine origin

The adrenal gland provides an important function by integrating neuronal, immune, vascular, metabolic and endocrine signals under a common organ capsule. It is the central organ of the stress response system and has been implicated in numerous stress-related disorders. While for other diseases, regeneration of healthy organ tissue has been aimed at such approaches are lacking for endocrine diseases - with the exception of type-I-diabetes. Moreover, adrenal tumor formation is very common, however, appropriate high-throughput applications reflecting the high heterogeneity and furthermore relevant 3D-structures in vitro are still widely lacking. Recently, we have initiated the development of standardized multidimensional models of a variety of endocrine cell/tissue sources in a new multiwell-format. Firstly, we confirmed common applicability for pancreatic pseudo-islets. Next, we translated applicability for spheroid establishment to adrenocortical cell lines as well as patient material to establish spheroids from malignant, but also benign adrenal tumors. We aimed furthermore at the development of bovine derived healthy adrenal organoids and were able to establish steroidogenic active organoids containing both, cells of cortical and medullary origin. Overall, we hope to open new avenues for basic research, endocrine cancer and adrenal tissue-replacement-therapies as we demonstrate potential for innovative mechanistic insights and personalized medicine in endocrine (tumor)-biology

Recurrent Disease in Patients With Sporadic Pheochromocytoma and Paraganglioma

Supplemental Appendix for manuscript 'Recurrent disease in patients with sporadic pheochromocytoma and paraganglioma

PD-L1 and HIF-2α Upregulation in Head and Neck Paragangliomas after Embolization

Hypoxia activates pathways associated with tumor progression, metastatic spread, and alterations in the immune microenvironment leading to an immunosuppressive phenotype. In particular, the upregulation of PD-L1, a target for therapy with checkpoint inhibitors, is well-studied in several tumors. However, the relationship between hypoxia and PD-L1 regulation in pheochromocytomas and paragangliomas (PPGL), and especially in paragangliomas treated with embolization, is still largely unexplored. We investigated the expression of the hypoxia-marker HIF-2α and of PD-L1 in a PPGL-cohort with and without embolization as potential biomarkers that may predict the response to treatment with HIF-2α and checkpoint inhibitors. A total of 29 tumor samples from 25 patients who were operated at a single center were included and analyzed utilizing immunohistochemistry (IHC) for PD-L1 and HIF-2α. Embolization prior to surgery was performed in seven (24%) tumors. PD-L1 expression in tumor cells of head and neck paragangliomas (HNPGLs) receiving prior embolization (median PD-L1 positivity: 15%) was significantly higher as compared to PD-L1 expression in HNPGLs without prior embolization (median PD-L1 positivity: 0%) (p = 0.008). Consistently, significantly more HNPGLs with prior embolization were positive for HIF-2α (median nuclear HIF-2α positivity: 40%) as compared to HNPGLs without prior embolization (median nuclear HIF-2α positivity: 0%) (p = 0.016). Our results support the hypothesis that embolization with subsequent hypoxia leads to the upregulation of both PD-L1 and HIF-2α in HNPGLs, and could thus facilitate targeted treatment with HIF-2α and checkpoint inhibitors in the case of inoperable, locally advanced, or metastatic disease

Proneuropeptide Y and neuropeptide Y metabolites in healthy volunteers and patients with a pheochromocytoma or paraganglioma

Neuropeptide Y (NPY1-36) is a vasoconstrictor peptide co-secreted with catecholamines by sympathetic nerves, the adrenal medulla, and neoplasms such as pheochromocytomas and paragangliomas (PPGLs). It is produced by the intracellular cleavage of proNPY and metabolized into multiple fragments with distinct biological activities. NPY immunoassays for PPGL have a diagnostic sensitivity ranging from 33 to 100%, depending on the antibody used. We have validated a multiplex micro-UHPLC-MS/MS assay for the specific and sensitive quantification of proNPY, NPY1-39, NPY1-37, NPY1-36, NPY2-36, NPY3-36, NPY1-35, NPY3-35, and the C-flanking peptide of NPY (CPON) (collectively termed NPYs), and determined the NPYs reference intervals and concentrations in 32 PPGL patients before, during, and after surgery. Depending on the peptide measured, NPYs were above the upper reference limit (URL) in 20% to 67% of patients, whereas plasma free metanephrine and normetanephrine, the gold standard for PPGL, were above the URL in 40% and 87% of patients, respectively. Age, sex, tachycardia, and tumor localization were not correlated with NPYs. Plasma free metanephrines performed better than NPYs in the detection of PPGL, but NPYs may be a substitute for an early diagnosis of PPGL for patients that suffer from severe kidney impairment or receiving treatments that interfere with catecholamine reuptake