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Hedgehog inhibitor sonidegib potentiates 177Lu-octreotate therapy of GOT1 human small intestine neuroendocrine tumors in nude mice.

ABSTRACT: 177Lu-octreotate can be used to treat somatostatin receptor expressing neuroendocrine tumors. It is highly effective in animal models, but clinical studies have so far only demonstrated low cure rates. Hedgehog inhibitors have shown therapeutic effect as monotherapy in neuroendocrine tumor model systems and might be one option to enhance the efficacy of 177Lu-octreotate therapy. The aim of this study was to determine the therapeutic effect of combination therapy using 177Lu-octreotate and the Hedgehog signaling pathway inhibitor sonidegib.GOT1-bearing BALB/c nude mice were treated with either sonidegib (80 mg/kg twice a week via oral gavage), a single injection of 30 MBq 177Lu-octreotate i.v., or a combination of both. Untreated animals served as controls. Tumor size was measured twice-weekly using calipers. The animals were killed 41 d after injection followed by excision of the tumors. Total RNA was extracted from each tumor sample and then subjected to gene expression analysis. Gene expression patterns were compared with those of untreated controls using Nexus Expression 3.0, IPA and Gene Ontology terms. Western blot was carried out on total protein extracted from the tumor samples to analyze activation-states of the Hh and PI3K/AKT/mTOR pathways.Sonidegib monotherapy resulted in inhibition of tumor growth, while a significant reduction in mean tumor volume was observed after 177Lu-octreotate monotherapy and combination therapy. Time to progression was prolonged in the combination therapy group compared with 177Lu-octreotate monotherapy. Gene expression analysis revealed a more pronounced response following combination therapy compared with both monotherapies, regarding the number of regulated genes and biological processes. Several cancer-related signaling pathways (i.e. Wnt/β-catenin, PI3K/AKT/mTOR, G-protein coupled receptor, and Notch) were affected by the combination therapy, but not by either monotherapy. Protein expression analysis revealed an activation of the Hh- and PI3K/AKT/mTOR pathways in tumors exposed to 177Lu-octreotate monotherapy and combination therapy.A comparative analysis of the different treatment groups showed that combination therapy using sonidegib and 177Lu-octreotate could be beneficial to patients with neuroendocrine tumors. Gene expression analysis revealed a functional interaction between sonidegib and 177Lu-octreotate, i.e. several cancer-related signaling pathways were modulated that were not affected by either monotherapy. Protein expression analysis indicated a possible PI3K/AKT/mTOR-dependent activation of the Hh pathway, independent of SMO.

SUBMITTER: Spetz J

PROVIDER: S-EPMC5549301 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Project description:177Lu‑DOTATATE was recently approved for the treatment of somatostatin receptor (SSTR)‑positive neuroendocrine 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.

Project description:BACKGROUND:177Lu-octreotate is used for therapy of somatostatin receptor expressing neuroendocrine tumors with promising results, although complete tumor remission is rarely seen. Previous studies on nude mice bearing the human small intestine neuroendocrine tumor, GOT1, have shown that a priming injection of 177Lu-octreotate 24 h before the main injection of 177Lu-octreotate resulted in higher 177Lu concentration in tumor, resulting in increased absorbed dose, volume reduction, and time to regrowth. To our knowledge, the cellular effects of a priming treatment schedule have not yet been studied. The aim of this study was to identify transcriptional changes contributing to the enhanced therapeutic response of GOT1 tumors in nude mice to 177Lu-octreotate therapy with priming, compared with non-curative monotherapy. RESULTS:RNA microarray analysis was performed on tumor samples from GOT1-bearing BALB/c nude mice treated with a 5 MBq priming injection of 177Lu-octreotate followed by a second injection of 10 MBq of 177Lu-octreotate after 24 h and killed after 1, 3, 7, and 41 days after the last injection. Administered activity amounts were chosen to be non-curative, in order to facilitate the study of tumor regression and regrowth. Differentially regulated transcripts (RNA samples from treated vs. untreated animals) were identified (change ≥ 1.5-fold; adjusted p value < 0.01) using Nexus Expression 3.0. Analysis of the biological effects of transcriptional regulation was performed using the Gene Ontology database and Ingenuity Pathway Analysis. Transcriptional analysis of the tumors revealed two stages of pathway regulation for the priming schedule (up to 1 week and around 1 month) which differed distinctly from cellular responses observed after monotherapy. Induction of cell cycle arrest and apoptotic pathways (intrinsic and extrinsic) was found at early time points after treatment start, while downregulation of pro-proliferative genes were found at a late time point. CONCLUSIONS:The present study indicates increased cellular stress responses in the tumors treated with a priming treatment schedule compared with those seen after conventional 177Lu-octreotate monotherapy, resulting in a more profound initiation of cell cycle arrest followed by apoptosis, as well as effects on PI3K/AKT-signaling and unfolded protein response.

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Hedgehog inhibitor sonidegib potentiates 177Lu-octreotate therapy of GOT1 human small intestine neuroendocrine tumors in nude mice.

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