NCI-H727 Cells Research Articles

Applying HDACis to increase SSTR2 expression and radiolabeled DOTA-TATE uptake: from cells to mice

AimsThe aim of our study was to determine the effect of histone deacetylase (HDAC) inhibitors (HDACis) on somatostatin type-2 receptor (SSTR2) expression and [111In]In-/[177Lu]Lu-DOTA-TATE uptake in vitro and in vivo. Materials and methodsThe human cell lines NCI-H69 (small-cell lung carcinoma) and BON-1 (pancreatic neuroendocrine tumor) were treated with HDACis (i.e. entinostat, mocetinostat (MOC), LMK-235, CI-994 or panobinostat (PAN)), and SSTR2 mRNA expression levels and [111In]In-DOTA-TATE uptake were measured. Furthermore, vehicle- and HDACi-treated NCI-H69 and BON-1 tumor-bearing mice were injected with radiolabeled DOTA-TATE followed by biodistribution studies. Additionally, SSTR2 and HDAC mRNA expression of xenografts, and of NCI-H69, BON-1, NCI-H727 (human pulmonary carcinoid) and GOT1 (human midgut neuroendocrine tumor) cells were determined. Key findingsHDACi treatment resulted in the desired effects in vitro. However, no significant increase in tumoral DOTA-TATE uptake was observed after HDACi treatment in NCI-H69 tumor-bearing animals, whereas tumoral SSTR2 mRNA and/or protein expression levels were significantly upregulated after treatment with MOC, CI-994 and PAN, i.e. a maximum of 2.1- and 1.3-fold, respectively. Analysis of PAN-treated BON-1 xenografts solely demonstrated increased SSTR2 mRNA expression levels. Comparison of HDACs and SSTR2 expression in BON-1 and NCI-H69 xenografts showed a significantly higher expression of 6/11 HDACs in BON-1 xenografts. Of these HDACs, a significant inverse correlation was found between HDAC3 and SSTR2 expression (Pearson r = −0.92) in the studied cell lines. SignificanceTo conclude, tumoral uptake levels of radiolabeled DOTA-TATE were not enhanced after HDACi treatment in vivo, but, depending on the applied inhibitor, increased SSTR2 expression levels were observed.

Cabozantinib in neuroendocrine tumors: tackling drug activity and resistance mechanisms.

Neuroendocrine tumors (NETs) are highly vascularized malignancies in which angiogenesis may entail cell proliferation and survival. Among the emerging compounds with antivascular properties, cabozantinib (CAB) appeared promising. We analyzed the antitumor activity of CAB against NETs utilizing in vitro and in vivo models. For cell cultures, we used BON-1, NCI-H727 and NCI-H720 cell lines. Cell viability was assessed by manual count coupled with quantification of cell death, performed through fluorescence-activated cell sorting analysis as propidium iodide exclusion assay. In addition, we investigated the modulation of the antiapoptotic myeloid cell leukemia 1 protein under CAB exposure, as a putative adaptive pro-survival mechanism, and compared the responses with sunitinib. The activity of CAB was also tested in mouse and zebrafish xenograft tumor models. Cabozantinib showed a dose-dependent and time-dependent effect on cell viability and proliferation in human NET cultures, besides a halting of cell cycle progression for endoduplication, never reported for other tyrosine kinase inhibitors. In a transplantable zebrafish model, CAB drastically inhibited NET-induced angiogenesis and migration of implanted cells through the embryo body. CAB showed encouraging activity in NETs, both in vitro and in vivo models. On this basis, we envisage future research to further investigate along these promising lines.

In Vitro Radioenhancement Using Ultrasound-Stimulated Microbubbles: A Comparison of Suspension and Adherent Cell States

Background: Ultrasound-stimulated microbubbles (USMB) have shown potential for enhancing radiation treatment via cavitation and sonoporation mechanisms. However, in vitro studies have produced inconsistent results, with adherent cells demonstrating no radioenhancement. This study aims to investigate the effect of cell adherence on in vitro radioenhancement using USMB and radiation. Method: Lung metastases of follicular thyroid carcinoma cells (FTC-238) and non-small cell lung carcinoma cells (NCI-H727) were treated, both when adhered and in suspension, using 1.6% (v/v) Definity™ microbubbles, ~90 s of 2 MHz ultrasound with mechanical index 0.9, and either 3 Gy or 6 Gy of megavoltage (MV) X-rays. The cell viability was measured using an MTS assay 72 h post-treatment, and statistical analysis was conducted using a three-way analysis of variance. Results: Statistically significant differences were observed for cells treated when adherent compared to suspended. An additive effect was detected in NCI-H727 cells treated in suspension, but not while adherent, while no enhancement was observed for FTC-238 cells in either culture state. Conclusions: To the best of our knowledge, this is the first study to directly compare the effect of cell adherence on the radioenhancement potential of USMB in vitro, and the first to do so using a metastatic cell line.

Ultrasound-Stimulated Microbubbles Enhance Radiation-Induced Cell Killing.

Recent in vivo studies using ultrasound-stimulated microbubbles as a localized radiosensitizer have had impressive results. While in vitro studies have also obtained similar results using human umbilical vein endothelial cells (HUVEC), studies using other cell lines have had varying results. This study was aimed at investigating any increases in radiation-induced cell killing in vitro using two carcinoma lines not previously investigated before (metastatic follicular thyroid carcinoma cells [FTC-238] and non-small cell lung carcinoma cells [NCI-H727]), in addition to HUVEC. Cells were treated using a combination of 1.6% (v/v) microbubbles, ∼90 s of 2-MHz ultrasound (mechanical index=0.8) and 0-6 Gy of kilovolt or MV X-rays. Cell viability assays obtained 72 h post-treatment were normalized to untreated controls, and analysis of variance was used to determine statistical significance. All cells treated with combined ultrasound-stimulated microbubbles and radiation exhibited decreased normalized survival, with statistically significant effects observed for the NCI-H727 cells. No statistically significant differences in effects were observed using kV compared with MV radiation. Further studies using increased microbubble concentrations may be required to achieve statistically significant results for the FTC-238 and HUVEC lines.

Cytotoxic effect of cloned EGFP gene on NCI-H727 cell line via genetically engineered gene transfer system

Introduction and Aim: Cancers are a complex group of genetic illnesses that develop through multistep, mutagenic processes which can invade or spread throughout the body. Recent advances in cancer treatment involve oncolytic viruses to infect and destroy cancer cells. The Newcastle disease virus (NDV), an oncolytic virus has shown to have anti-cancer effects either directly by lysing cancer cells or indirectly by activating the immune system. The green fluorescent protein (GFP) has been widely used in studying the anti-tumor activity of oncolytic viruses. This study aimed to study the anticancer effect of a recombinant rNDV-GFP clone on NCI-H727 lung carcinoma cell line in vitro. Materials and Methods: The GFP gene was inserted to a NDV strain to create a recombinant NDV (rNDV- GFP) using reverse genetics technology. The MTT assay was used in evaluating the oncolytic effect of rNDV- GFP on the lung carcinoma NCI-H727 cells. Light and fluorescent microscopy was used to study the cytopathic effects of rNDV-GFP. Results: MTT assay showed that rNDV-GPF inhibited the NCI-H727 tumor cell death in a time-dependent manner. A significant inhibitory effect (78.3%) for rNDV-GPF on cancer cells was observed at 96h in comparison to rNDV (22.7%) and the cytotoxicity rate was directly proportional to the MOI used. Microscopic studies showed rNDV-GPF to induce cytopathic effect post 24 h of infection. Conclusion: The GFP-expressing recombinant NDV strains exhibited encouraging results in terms of tumor growth inhibition. Our research set the groundwork for employing recombinant NDV as an anticancer viral vector.

Comparing the Effect of Multiple Histone Deacetylase Inhibitors on SSTR2 Expression and [111In]In-DOTATATE Uptake in NET Cells.

Simple SummaryPatients diagnosed with neuroendocrine tumors (NETs) are often treated with peptide receptor radionuclide therapy (PRRT). This therapy targets the somatostatin type-2 receptors (SSTR2) frequently overexpressed on these types of tumors. Although this therapy has proven to be effective, complete responses are rare and therapy improvement is desirable. We aimed to increase SSTR2 expression on NET cells, potentially increasing the number of patients eligible for SSTR2-targeted PRRT and improving clinical outcomes. We used histone deacetylase inhibitors (HDACis) to manipulate the epigenetic machinery and hereby aimed to increase SSTR2 gene transcription. Our results showed that the HDACis increased SSTR2 expression in several NET cell lines. Moreover, the uptake of radiolabeled DOTATATE, the tracer used for PRRT, was enhanced. The observed reversibility profile after HDACi withdrawal of the induced effects suggests that proper timing of HDACi treatment is likely essential.The aim of this study was to increase somatostatin type-2 receptor (SSTR2) expression on neuroendocrine tumor (NET) cells using histone deacetylase inhibitors (HDACis), potentially increasing the uptake of SSTR2-targeted radiopharmaceuticals and subsequently improving treatment efficacy of peptide receptor radionuclide therapy (PRRT). Human NET cell lines BON-1, NCI-H727, and GOT1 were treated with HDACis (i.e., CI-994, entinostat, LMK-235, mocetinostat, panobinostat, or valproic acid (VPA); entinostat and VPA were the HDACis tested in GOT1 cells) to examine SSTR2 mRNA expression levels and uptake of SSTR2-targeting radiotracer [111In]In-DOTATATE. Reversibility of the induced effects was examined after drug-withdrawal. Finally, the effect of VPA on radiosensitivity was investigated. A strong stimulatory effect in BON-1, NCI-H727, and GOT1 cells was observed after HDACi treatment, both on SSTR2 mRNA expression levels and [111In]In-DOTATATE uptake. The effects of the HDACis were largely reversible over a period of seven days, demonstrating largest reductions within the first day. The reversibility profile of the induced effects suggests that proper timing of HDACi treatment is most likely essential for a beneficial outcome. In addition to increasing SSTR2 expression levels, VPA enhanced the radiosensitivity of all cell lines. In conclusion, HDACi treatment increased SSTR2 expression, and radiosensitivity was also enhanced upon VPA treatment.

Antitumoral Activity of the MEK Inhibitor Trametinib (TMT212) Alone and in Combination with the CDK4/6 Inhibitor Ribociclib (LEE011) in Neuroendocrine Tumor Cells In Vitro.

Simple SummarySystemic treatment options for advanced neuroendocrine tumors have significantly been improved in the last decade. However efficacy of systemic therapy is limited by tumor resistance and therefore there is a need for further treatment options. Inhibition of the Ras-Raf-Mek-Erk signaling cascade might be a promising new treatment strategy in neuroendocrine neoplasms. In this study we investigated the effects of the MEK inhibitor trametinib, the ERK inhibitor SCH772984 and the CDK4/6 inhibitor ribociclib in human neuroendocrine tumor cell lines BON1, QGP1 and NCI-H727 in vitro. Trametinib alone and in synergism with ribociclib demonstrated antiproliferative effects. Combination therapy of MEK inhibitors and CDK4/6 inhibitors might be a potential strategy to overcome CDK4/6 inhibitor resistance in neuroendocrine tumors.Objectives: This study assessed the antitumoral activity of the MEK inhibitor trametinib (TMT212) and the ERK1/2 inhibitor SCH772984, alone and in combination with the CDK4/6 inhibitor ribociclib (LEE011) in human neuroendocrine tumor (NET) cell lines in vitro. Methods: Human NET cell lines BON1, QGP-1, and NCI-H727 were treated with trametinib or SCH772984, alone and in combination with ribociclib, to assess cell proliferation, cell cycle distribution, and protein signaling using cell proliferation, flow cytometry, and Western blot assays, respectively. Results: Trametinib and SCH772984, alone and in combination with ribociclib, significantly reduced NET cell viability and arrested NET cells at the G1 phase of the cell cycle in all three cell lines tested. In addition, trametinib also caused subG1 events and apoptotic PARP cleavage in QGP1 and NCI-H727 cells. A western blot analysis demonstrated the use of trametinib alone and trametinib in combination with ribociclib to decrease the expression of pERK, cMyc, Chk1, pChk2, pCDK1, CyclinD1, and c-myc in a time-dependent manner in NCI-H727 and QGP-1 cells. Conclusions: MEK and ERK inhibition causes antiproliferative effects in human NET cell lines in vitro. The combination of the MEK inhibitor trametinib (TMT212) with the CDK4/6 inhibitor ribociclib (LEE011) causes additive antiproliferative effects. Future preclinical and clinical studies of MEK inhibition in NETs should be performed.

Effects of fluorodeoxyglucose magnetic nanoparticles on NCI-H727 and SH-SY5Y cancer cells.

We present a report regarding the cytotoxic effects of iron-based magnetic nanoparticles conjugated with fluorodeoxyglucose (FDG-mNPs) on the viability of NCI-H727 and SH-SY5Y cancer cells. MTT assays were performed to determine cell viability in treated cancer cells grown under standard 2D culture conditions. FDG-mNP concentrations of 0.075 mg/mL, 0.15 mg/mL, and 0.3 mg/mL decreased mean cell viability of NCI-H727 cells to 92.5%, 82.9%, and 75% respectively. FDG-mNPs was also shown to have a detrimental effect on the viability of SY5Y cells: a decrease of 5.7%, 18.6%, and 36.4% was found for SY5Y cells treated with 0.075 mg/mL, 0.15 mg/mL, and 0.3 mg/mL concentrations of FDG-mNPs, respectively. When NCI-H727 and SH-SY5Y cancer cells were grown as 3D spheroids, morphology was visibly changed and the number of viable cells was decerased in spheroids treated with FDG-mNPs compared with untreated spheroids. The results of our study demonstrated that FDG-mNP has toxic effects on NCI-H7272 and SY5Y cancer cells, and we conclude that conjugated FDG-mNPs are promising in the development of clinical applications for the destruction of cancer cells.

Multiple modes of cell death in neuroendocrine tumors induced by artesunate

BackgroundThe paucity of effective treatment in neuroendocrine tumors (NETs) encouraged us to investigate the therapeutic value of artesunate (ART) promised by its inhibitory effect against various tumors and broad safety profile. MethodsWe evaluated the impact of ART on three NET cell lines, BON-1, QGP-1 and NCI-H727 on cellular and molecular levels. ResultsOur results showed that ART induced endoplasmic reticulum (ER) stress through phosphorylation of eIF2α, which further gave rise to autophagy in all three NET cell lines. Specifically, apoptosis and ferroptosis were also observed in BON-1 cells, which made BON-1 cell line more vulnerable upon ART treatment. The different sensitivities presented on the three cell lines also associated with a differential regulation of p21 on the long run. Co-treatment with p21 inhibitor UC2288 showed an additive effect on QGP-1 and NCI-H727 cell lines indicating p21 upregulation in these two cell lines might confer resistance towards ART treatment. ConclusionsIt is possible to include ART in the treatment of NETs in the future.

Inhibition of Wnt/β-Catenin Signaling in Neuroendocrine Tumors in vitro: Antitumoral Effects.

Background and aims: Inhibition of Wnt/β-catenin signaling by specific inhibitors is currently being investigated as an antitumoral strategy for various cancers. The role of Wnt/β-catenin signaling in neuroendocrine tumors still needs to be further investigated. Methods: This study investigated the antitumor activity of the porcupine (PORCN) inhibitor WNT974 and the β-catenin inhibitor PRI-724 in human neuroendocrine tumor (NET) cell lines BON1, QGP-1, and NCI-H727 in vitro. NET cells were treated with WNT974, PRI-724, or small interfering ribonucleic acids against β-catenin, and subsequent analyses included cell viability assays, flow cytometric cell cycle analysis, caspase3/7 assays and Western blot analysis. Results: Treatment of NET cells with WNT974 significantly reduced NET cell viability in a dose- and time-dependent manner by inducing NET cell cycle arrest at the G1 and G2/M phases without inducing apoptosis. WNT974 primarily blocked Wnt/β-catenin signaling by the dose- and time-dependent downregulation of low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation and non-phosphorylated β-catenin and total β-catenin, as well as the genes targeting the latter (c-Myc and cyclinD1). Furthermore, the WNT974-induced reduction of NET cell viability occurred through the inhibition of GSK-3-dependent or independent signaling (including pAKT/mTOR, pEGFR and pIGFR signaling). Similarly, treatment of NET cells with the β-catenin inhibitor PRI-724 caused significant growth inhibition, while the knockdown of β-catenin expression by siRNA reduced NET tumor cell viability of BON1 cells but not of NCI-H727 cells. Conclusions: The PORCN inhibitor WNT974 possesses antitumor properties in NET cell lines by inhibiting Wnt and related signaling. In addition, the β-catenin inhibitor PRI-724 possesses antitumor properties in NET cell lines. Future studies are needed to determine the role of Wnt/β-catenin signaling in NET as a potential therapeutic target.

Metformin Inhibits Proliferation and Tumor Growth of QGP-1 Pancreatic Neuroendocrine Tumor Cells by Inducing Cell Cycle Arrest and Apoptosis.

Pancreatic neuroendocrine tumors (pNETs) are rare pancreatic neoplasms, and therapeutic options for pNETs are limited. Metformin is an anti-hypoglycemic drug that appears to have anticancer effects. However, little is known about the effect of metformin on pNETs. In this study, we investigated the anti-proliferative effect of metformin on a human pNET cell line. The anti-proliferative properties of metformin were evaluated in QGP-1 and NCI-H727 cells using a cell counting kit-8 assay. Xenograft mouse models were used to assess the tumor effect in vivo. Metformin inhibited the proliferation and anti-tumor growth of QGP-1 cells, accompanied by their arrest during the cell cycle at the G0/G1 phase. Immunohistochemical analysis of tumor tissues revealed down-regulation of cyclin D1 and proliferating cell nuclear antigen in the metformin-treated group. Additionally, metformin induced apoptosis, and the expression of survivin and claspin were decreased in metformin-treated QGP-1 cells according to the apoptosis array. Furthermore, the angiogenic related protein TIMP-1 was down-regulated, and its miRNA expression was altered by metformin in QGP-1 cells. Taken together, our study demonstrated the therapeutic potential of metformin and provides molecular mechanistic insights into its anti-tumoral effect on pNETs. This study is the first one describing anti-tumoral effects in pNETs.

The HSP90 inhibitor onalespib potentiates 177Lu‑DOTATATE therapy in neuroendocrine tumor cells.

177Lu-DOTATATE was recently approved for the treatment of somatostatin receptor (SSTR)-positive neuroen-docrine tumors (NETs). However, despite impressive response rates, complete responses are rare. Heat shock protein 90 (HSP90) inhibitors have been suggested as suitable therapeutic agents for NETs, as well as a potential radiosensitizers. Consequently, the aim of this study was to investigate whether the HSP90-inhibitor onalespib could reduce NET cell growth and act as a radiosensitizer when used in combination with 177Lu-DOTATATE. The NET cell lines BON, NCI-H727 and NCI-H460, were first characterized with regards to 177Lu-DOTATATE uptake and sensitivity to onalespib treatment in monolayer cell assays. The growth inhibitory effects of the monotherapies and combination treatments were then examined in three-dimensional multicellular tumor spheroids. Lastly, the molecular effects of the treatments were assessed. 177Lu-DOTATATE uptake was observed in the BON and NCI-H727 cells, while the NCI-H460 cells exhibited no detectable uptake. Accordingly, 177Lu-DOTATATE reduced the growth of BON and NCI-H727 spheroids, while no effect was observed in the NCI-H460 spheroids. Onalespib reduced cell viability and spheroid growth in all three cell lines. Furthermore, the combination of onalespib and 177Lu-DOTATATE exerted synergistic therapeutic effects on the BON and NCI-H727 spheroids. Western blot analysis of BON spheroids revealed the downregulation of epidermal growth factor receptor (EGFR) and the upregulation of γ H2A histone family member X (γH2AX) following combined treatment with onalespib and 177Lu-DOTATATE. Moreover, flow cytometric analyses revealed a two-fold increase in caspase 3/7 activity in the combination group. In conclusion, the findings of this study demonstrate that onalespib exerts antitumorigenic effects on NET cells and may thus be a feasible treatment option for NETs. Furthermore, onalespib was able to synergistically potentiate 177Lu-DOTATATE treatment in a SSTR-specific manner. The radiosensitizing mechanisms of onalespib involved the downregulation of EGFR expression and the induction of apoptosis. Consequently, the combination of onalespib and 177Lu-DOTATATE may prove to be a promising strategy with which to improve therapeutic responses in patients with NETs. Further studies investigating this strategy in vivo regarding the therapeutic effects and potential toxicities are warranted to expand these promising findings.

Somatostatin Analogue Treatment Primarily Induce miRNA Expression Changes and Up-Regulates Growth Inhibitory miR-7 and miR-148a in Neuroendocrine Cells

Somatostatin (SST) analogues are used to control the proliferation and symptoms of neuroendocrine tumors (NETs). MicroRNAs (miRNA) are small non-coding RNAs that modulate posttranscriptional gene expression. We wanted to characterize the miRNAs operating under the control of SST to elucidate to what extent they mediate STT actions. NCI-H727 carcinoid cell line was treated with either a chimeric SST/dopamine analogue; a SST or dopamine analogue for proliferation assays and for identifying differentially expressed miRNAs using miRNA microarray. The miRNAs induced by SST analogue treatment are investigated in carcinoid cell lines NCI-H727 and CNDT2 using in situ hybridization, qPCR and proliferation assays. SST analogues inhibited the growth of carcinoid cells more potently compared to the dopamine analogue. Principal Component Analysis (PCA) of the samples based on miRNA expression clearly separated the samples based on treatment. Two miRNAs which were highly induced by SST analogues, miR-7 and miR-148a, were shown to inhibit the proliferation of NCI-H727 and CNDT2 cells. SST analogues also produced a general up-regulation of the let-7 family members. SST analogues control and induce distinct miRNA expression patterns among which miR-7 and miR-148a both have growth inhibitory properties.

Abstract 5398: Companion diagnostic for predicting the efficacy of mTOR inhibitor in neuroendocrine tumor

Abstract Neuroendocrine tumor (NET) is having both characterizations of hormone and neurotransmitter secretion. The mTOR, a serine-threonine kinase inhibitor, was used for treating the NET tumors by affecting angiogeneic blood vascular vessels as a disrupting agent and killing the tumor cells by inhibiting the mTOR signalling. Everolimus is a U.S. FDA-approved drug for treating advanced breast cancer, which is investigated for use in NET treatment. The efficacy will be evaluated by exosome reflecting the tumor microenvironment secreted into blood circulation. NCI-H727 cells are a kind of NET tumor that show the secretion of chromogranin A (CgA) and serotonin. By profiling of exosome with miRNA, the efficacy may be predicted by the level of miRNA. Here, we demonstrated the pattern of miRNA profile in everolimus-treated NCI-H727 cells. MIR192 and MIR375 were consistently secreted in exosomes with a high level. A panel of miRNA, MIR24-2, MIR3131, and MIR708, was increased and showed a change greater than 0.5 fold compared with untreated cells. In conclusion, by analyzing the pattern of miRNA in exosomes, the efficacy of drug can be monitored during the treatment. A new therapeutic strategy can be tailored according to the miRNA pattern. Citation Format: I-Hua Liu, Jia-Ming Chang. Companion diagnostic for predicting the efficacy of mTOR inhibitor in neuroendocrine tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5398.

Tropomyosin receptor kinase: a novel target in screened neuroendocrine tumors.

Tropomyosin receptor kinase (Trk) inhibitors are investigated as a novel targeted therapy in various cancers. We investigated the in vitro effects of the pan-Trk inhibitor GNF-5837 in human neuroendocrine tumor (NET) cells. The human neuroendocrine pancreatic BON1, bronchopulmonary NCI-H727 and ileal GOT1 cell lines were treated with GNF-5837 alone and in combination with everolimus. Cell viability decreased in a time- and dose-dependent manner in GOT1 cells in response to GNF-5837 treatment, while treatment in BON1 and NCI-H727 cells showed no effect on cellular viability. Trk receptor expression determined GNF-5837 sensitivity. GNF-5837 caused downregulation of PI3K-Akt-mTOR signaling, Ras-Raf-MEK-ERK signaling, the cell cycle and increased apoptotic cell death. The combinational treatment of GNF-5837 with everolimus showed a significant enhancement in inhibition of cell viability vs single substance treatments, due to a cooperative PI3K-Akt-mTOR and Ras-Raf-MEK-ERK pathway downregulation, as well as an enhanced cell cycle component downregulation. Immunohistochemical staining for Trk receptors were performed using a tissue microarray containing 107 tumor samples of gastroenteropancreatic NETs. Immunohistochemical staining with TrkA receptor and pan-Trk receptor antibodies revealed a positive staining in pancreatic NETs in 24.2% (8/33) and 33.3% (11/33), respectively. We demonstrated that the pan-Trk inhibitor GNF-5837 has promising anti-tumoral properties in human NET cell lines expressing the TrkA receptor. Immunohistochemical or molecular screening for Trk expression particularly in pancreatic NETs might serve as predictive marker for molecular targeted therapy with Trk inhibitors.

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 HDM2 (MDM2) Inhibitor NVP-CGM097 Inhibits Tumor Cell Proliferation and Shows Additive Effects with 5-Fluorouracil on the p53-p21-Rb-E2F1 Cascade in the p53wild type Neuroendocrine Tumor Cell Line GOT1

Background/Aims: The tumor suppressor p53 is depleted in many tumor cells by the E3 ubiquitin ligase mouse double minute 2 homolog (MDM2) through MDM2/p53 interaction. A novel target for inhibiting p53 degradation and for causing reexpression of p53^{wild type} is inhibition of MDM2. The small molecule NVP-CGM097 is a novel MDM2 inhibitor. We investigated MDM2 inhibition as a target in neuroendocrine tumor cells in vitro. Methods: Human neuroendocrine tumor cell lines from the pancreas (BON1), lung (NCI-H727), and midgut (GOT1) were incubated with the MDM2 inhibitor NVP-CGM097 (Novartis) at concentrations from 4 to 2,500 n<smlcap>M</smlcap>. Results: While p53^{wild type} GOT1 cells were sensitive to NVP-CGM097, p53^mutated BON1 and p53^mutated NCI-H727 cells were resistant to NVP-CGM097. Incubation of GOT1 cells with NVP-CGM097 at 100, 500, and 2,500 n<smlcap>M</smlcap> for 96 h caused a significant decline in cell viability to 84.9 ± 9.2% (p < 0.05), 77.4 ± 6.6% (p < 0.01), and 47.7 ± 9.2% (p < 0.01). In a Western blot analysis of GOT1 cells, NVP-CGM097 caused a dose-dependent increase in the expression of p53 and p21 tumor suppressor proteins and a decrease in phospho-Rb and E2F1. Experiments of co-incubation of NVP-CGM097 with 5-fluorouracil, temozolomide, or everolimus each showed additive antiproliferative effects in GOT1 cells. NVP-CGM097 and 5-fluorouracil increased p53 and p21 expression in an additive manner. Conclusions: MDM2 inhibition seems a promising novel therapeutic target in neuroendocrine tumors harboring p53^{wild type}. Further investigations should examine the potential role of MDM2 inhibitors in neuroendocrine tumor treatment.

Effects of the single and combined treatment with dopamine agonist, somatostatin analog and mTOR inhibitors in a human lung carcinoid cell line: an in vitro study.

Somatostatin analogues and mTOR inhibitors have been used as medical therapy in lung carcinoids with variable results. No data are available on dopamine agonists as treatment for lung carcinoids. The main aim of the current study was to evaluate the effect of the combined treatment of somatostatin analogue octreotide and the dopamine agonist cabergoline with mTOR inhibitors in an in vitro model of typical lung carcinoids: the NCI-H727 cell line. In NCI-H727 cell line, reverse transcriptase-quantitative polymerase chain reaction and immunofluorescence were assessed to characterize the expression of the somatostatin receptor 2 and 5, dopamine receptor 2 and mTOR pathway components. Fifteen typical lung carcinoids tissue samples have been used for somatostatin receptor 2, dopamine receptor 2, and the main mTOR pathway component p70S6K expression and localization by immunohistochemistry. Cell viability, fluorescence-activated cell sorting analysis and western blot have been assessed to test the pharmacological effects of octreotide, cabergoline and mTOR inhibitors, and to evaluate the activation of specific cell signaling pathways in NCI-H727 cell line. NCI-H727 cell line expressed somatostatin receptor 2, somatostatin receptor 5 and dopamine receptor 2 and all mTOR pathway components at messenger and protein levels. Somatostatin receptor 2, dopamine receptor 2, and p70S6K (non phosphorylated and phosphorylated) proteins were expressed in most typical lung carcinoids tissue samples. Octreotide and cabergoline did not reduce cell viability as single agents but, when combined with mTOR inhibitors, they potentiate mTOR inhibitors effect after long-term exposure, reducing Akt and ERK phosphorylation, mTOR escape mechanisms, and increasing the expression DNA-damage-inducible transcript 4, an mTOR suppressor. In conclusion, the single use of octreotide and cabergoline is not sufficient to block cell viability but the combined approach of these agents with mTOR inhibitors might reduce the mTOR inhibitors-induced escape mechanisms and/or activate the endogenous mTOR suppressor, potentiating the effect of the mTOR inhibitors in an in vitro model of typical lung carcinoids.

Abstract 3482: Inhibition of glucose metabolism in neuroendocrine tumors by lanreotide, sunitinib, and rapamycin

Abstract Background: Neuroendocrine tumors (NETs) represent a heterogeneous family of neoplasms, which may develop from different endocrine glands or endocrine cells dispersed throughout the digestive and respiratory tracts. Lanreotide is a somatostatin analog approved for the treatment of NETs. While Lanreotide extends survival of patients and slows tumor growth, controversy exists regarding its mechanism of action. We hypothesized that lanreotide can interfere with the metabolic activity of NETs, and compared its effect to that of rapamycin and sunitinib. Methods: The pancreatic carcinoid BON-1 cells and pulmonary carcinoid NCI-H727 cells were used. Viability was tested using colony formation assay. mRNA levels of the glycolytic enzymes hexokinase 2 (HK2), lactate dehydrogenase (LDH) and pyruvate kinase M2 (PKM2) was tested using real-time PCR. Phosphorylation levels of AMP-activated kinase (AMPK) and acetyl CoA carboxylase (ACC) were tested using Western blot. The activity of HK2 was tested using HK enzymatic assay. Lactate secretion was tested in the routine biochemistry laboratory of our institution. Results: Lanreotide, rapamycin and sunitinib significantly decreased colony formation. They also reduced relative expression levels of HK2, LDH and PKM2 24 and 48h after treatment and decreased HK2 activity after 24h. An increase in the phosphorylation levels of AMPK and ACC was observed shortly after treating the cells with lanreotide and rapamycin. All three agents also decreased lactate concentration in the media. Conclusions: These data indicate tumor metabolism as an attractive target for therapies against NET, and a novel mechanism of action of somatostatin analogs, namely, alteration of tumor metabolism. Citation Format: Tami Rubinek, Ayelet Shabtay-Orbach, Arkadi Hesin, Tammi Rubinstein, Ido Wolf. Inhibition of glucose metabolism in neuroendocrine tumors by lanreotide, sunitinib, and rapamycin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3482.

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 μ<smlcap>M</smlcap>) 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.