Preclinical in vitro investigation to evaluate novel molecular targeted therapy options for neuroendocrine neoplasms

GEP-NETs are highly complex, rare and heterogeneous tumor entities [1]. Current treatment approaches are unsatisfying, due to their limited efficacy [2-4]. Ergo, novel therapeutic strategies, including molecularly targeted therapeutics, are urgently needed [9, 14]. The aim of this doctoral thesis was to evaluate different novel molecular targeted therapy options for neuroendocrine neoplasms in vitro. By targeting different signaling pathways, we obtained novel insights into NET tumor biology and assessed possible novel treatment strategies. Several dual-targeting approaches prevented feed-back loops within a signaling pathway, as well as cross-talk activation between signaling pathways and enhanced single substance treatment. This cumulative doctoral thesis is based on the following original publications: The novel cyclin-dependent kinase 4/6 inhibitor ribociclib (LEE011) alone and in dual -targeting approaches demonstrates antitumoral efficacy in neuroendocrine tumors in vitro; Neuroendocrinology. 2017 Feb; doi: 10.1159/000463386. PMID: 28226315. Aristizabal Prada ET, Nölting S, Spöttl G, Maurer J, Auernhammer CJ. The MTH1 inhibitor TH588 demonstrates anti-tumoral effects alone and in combination with everolimus, 5-FU and gamma-irradiation in neuroendocrine tumor cells. PLoS ONE. 2017 May; doi: 10.1371/journal.pone.0178375. PMID: 28542590. Aristizabal Prada ET, Orth M, Nölting S, Spöttl G, Maurer J, Auernhammer CJ

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 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

Primary Aldosteronism: KCNJ5 Mutations and Adrenocortical Cell Growth

Aldosterone-producing adenomas with somatic mutations in the KCNJ5 G-protein-coupled inwardly rectifying potassium channel are a cause of primary aldosteronism. These mutations drive aldosterone excess, but their role in cell growth is undefined. Our objective was to determine the role of KCNJ5 mutations in adrenal cell proliferation and apoptosis. The Ki67 proliferative index was positively correlated with adenoma diameter in aldosterone-producing adenomas with a KCNJ5 mutation (r=0.435, P=0.007), a negative correlation was noted in adenomas with no mutation detected (r=-0.548, P=0.023). Human adrenocortical cell lines were established with stable expression of cumate-inducible wild-type or mutated KCNJ5. Increased cell proliferation was induced by low-level induction of KCNJ5-T158A expression compared with control cells (P=0.009), but increased induction ablated this difference. KCNJ5-G151R displayed no apparent proliferative effect, but KCNJ5-G151E and L168R mutations each resulted in decreased cell proliferation (difference P<0.0001 from control cells, both comparisons). Under conditions tested, T158A had no effect on apoptosis, but apoptosis increased with expression of G151R (P<0.0001), G151E (P=0.008), and L168R (P<0.0001). We generated a specific KCNJ5 monoclonal antibody which was used in immunohistochemistry to demonstrate strong KCNJ5 expression in adenomas without a KCNJ5 mutation and in the zona glomerulosa adjacent to adenomas irrespective of genotype as well as in aldosterone-producing cell clusters. Double immunofluorescence staining for KCNJ5 and CYP11B2 (aldosterone synthase) showed markedly decreased KCNJ5 immunostaining in CYP11B2-positive cells compared with CYP11B2-negative cells in aldosterone-producing adenomas with a KCNJ5 mutation. Together, these findings support the concept that cell growth effects of KCNJ5 mutations are determined by the expression level of the mutated channel

The selective PI3Kα inhibitor BYL719 as a novel therapeutic option for neuroendocrine tumors: Results from multiple cell line models

BACKGROUND/AIMS The therapeutic options for metastatic neuroendocrine tumors (NETs) are limited. As PI3K signaling is often activated in NETs, we have assessed the effects of selective PI3Kp110\textgreeka inhibition by the novel agent BYL719 on cell viability, colony formation, apoptosis, cell cycle, signaling pathways, differentiation and secretion in pancreatic (BON-1, QGP-1) and pulmonary (H727) NET cell lines. METHODS Cell viability was investigated by WST-1 assay, colony formation by clonogenic assay, apoptosis by caspase3/7 assay, the cell cycle by FACS, cell signaling by Western blot analysis, expression of chromogranin A and somatostatin receptors 1/2/5 by RT-qPCR, and chromogranin A secretion by ELISA. RESULTS BYL719 dose-dependently decreased cell viability and colony formation with the highest sensitivity in BON-1, followed by H727, and lowest sensitivity in QGP-1 cells. BYL719 induced apoptosis and G0/G1 cell cycle arrest associated with increased p27 expression. Western blots showed inhibition of PI3K downstream targets to a varying degree in the different cell lines, but IGF1R activation. The most sensitive BON-1 cells displayed a significant, and H727 cells a non-significant, GSK3 inhibition after BYL719 treatment, but these effects do not appear to be mediated through the IGF1R. In contrast, the most resistant QGP-1 cells showed no GSK3 inhibition, but a modest activation, which would partially counteract the other anti-proliferative effects. Accordingly, BYL719 enhanced neuroendocrine differentiation with the strongest effect in BON-1, followed by H727 cells indicated by induction of chromogranin A and somatostatin receptor 1/2 mRNA-synthesis, but not in QGP-1 cells. In BON-1 and QGP-1 cells, the BYL719/everolimus combination was synergistic through simultaneous AKT/mTORC1 inhibition, and significantly increased somatostatin receptor 2 transcription compared to each drug separately. CONCLUSION Our results suggest that the agent BYL719 could be a novel therapeutic approach to the treatment of NETs that may sensitize NET cells to somatostatin analogs, and that if there is resistance to its action this may be overcome by combination with everolimus

Additive Anti-Tumor Effects of Lovastatin and Everolimus In Vitro through Simultaneous Inhibition of Signaling Pathways

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedThis work was supported by a research grant from the Ludwig-Maximilians University of Munich (Förderprogramm für Forschung und Lehre [FöFoLe], grant number 865/829)

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