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

Three Dimensional Models of Endocrine Organs and Target Tissues Regulated by the Endocrine System

Immortalized cell lines originating from tumors and cultured in monolayers in vitro display consistent behavior and response, and generate reproducible results across laboratories. However, for certain endpoints, these cell lines behave quite differently from the original solid tumors. Thereby, the homogeneity of immortalized cell lines and two-dimensionality of monolayer cultures deters from the development of new therapies and translatability of results to the more complex situation in vivo. Organoids originating from tissue biopsies and spheroids from cell lines mimic the heterogeneous and multidimensional characteristics of tumor cells in 3D structures in vitro. Thus, they have the advantage of recapitulating the more complex tissue architecture of solid tumors. In this review, we discuss recent efforts in basic and preclinical cancer research to establish methods to generate organoids/spheroids and living biobanks from endocrine tissues and target organs under endocrine control while striving to achieve solutions in personalized medicine

Improving susceptibility of neuroendocrine tumors to radionuclide therapies: personalized approaches towards complementary treatments

Radionuclide therapies are an important tool for the management of patients with neuroendocrine neoplasms (NENs). Especially [$^{131}$I]MIBG and [$^{177}$Lu]Lu-DOTA-TATE are routinely used for the treatment of a subset of NENs, including pheochromocytomas, paragangliomas and gastroenteropancreatic tumors. Some patients suffering from other forms of NENs, such as medullary thyroid carcinoma or neuroblastoma, were shown to respond to radionuclide therapy; however, no general recommendations exist. Although [$^{131}$I]MIBG and [$^{177}$Lu]Lu-DOTA-TATE can delay disease progression and improve quality of life, complete remissions are achieved rarely. Hence, better individually tailored combination regimes are required. This review summarizes currently applied radionuclide therapies in the context of NENs and informs about recent advances in the development of theranostic agents that might enable targeting subgroups of NENs that previously did not respond to [$^{131}$I]MIBG or [$^{177}$Lu]Lu-DOTA-TATE. Moreover, molecular pathways involved in NEN tumorigenesis and progression that mediate features of radioresistance and are particularly related to the stemness of cancer cells are discussed. Pharmacological inhibition of such pathways might result in radiosensitization or general complementary antitumor effects in patients with certain genetic, transcriptomic, or metabolic characteristics. Finally, we provide an overview of approved targeted agents that might be beneficial in combination with radionuclide therapies in the context of a personalized molecular profiling approach

The Vault Complex Is Significantly Involved in Therapeutic Responsiveness of Endocrine Tumors and Linked to Autophagy under Chemotherapeutic Conditions

Cancers display dynamic interactions with their complex microenvironments that influence tumor growth, invasiveness, and immune evasion, thereby also influencing potential resistance to therapeutic treatments. The tumor microenvironment (TME) includes cells of the immune system, the extracellular matrix, blood vessels, and other cell types, such as fibroblasts or adipocytes. Various cell types forming this TME secrete exosomes, and molecules thereby released into the TME have been shown to be important mediators of cellular communication and interplay. Specific stressors in the TME, such as hypoxia, starvation, inflammation, and damage, can furthermore induce autophagy, a fundamental cellular process that degrades and recycles molecules and subcellular components, and recently it has been demonstrated that the small non-coding vault RNA1-1 plays a role as a regulator of autophagy and the coordinated lysosomal expression and regulation (CLEAR) network. Here, we demonstrate for the first time that intra-tumoral damage following effective therapeutic treatment is linked to specific intracellular synthesis and subsequent exosomal release of vault RNAs in endocrine tumors in vitro and in vivo. While we observed a subsequent upregulation of autophagic markers under classical chemotherapeutic conditions, a downregulation of autophagy could be detected under conditions strongly involving inflammatory cascades

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