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The mechanistic target of rapamycin complex 1 (mTORC1) inhibitor everolimus is one of the few approved therapies for locally advanced and metastatic neuroendocrine tumours (NETs). However, after initial disease stabilisation, most patients develop resistance within 1 year. Our aim was to overcome resistance to everolimus by additional treatment with the PI3K-alpha inhibitor alpelisib in an everolimus-resistant orthotopic pancreatic neuroendocrine carcinoma xenograft mouse model. Female SCID mice underwent laparoscopic pancreatic transplantation of everolimus-sensitive (BON1KDMSO) or everolimus-resistant (BON1RR2) NET cells. Both groups were further divided into four treatment groups: placebo, everolimus, alpelisib, and everolimus + alpelisib (combination). Oral treatment was started at a tumour volume of approximately 140 mm3 and continued until 1900-2000 mm3, validated by weekly MRI. Somatostatin receptor expression and tumour viability were analysed by 68Ga-DOTATOC and 18F-FDG PET/CT. Everolimus resistance of the BON1RR2 tumours was confirmed. In the everolimus-sensitive group, everolimus alone, alpelisib alone, and combination treatment significantly prolonged survival, compared to placebo, while in the BON1RR2 group, only combination treatment significantly prolonged survival compared to placebo, but neither everolimus nor alpelisib alone. Placebo-treated everolimus-sensitive tumours grew more rapidly (median survival 45 days), compared to placebo-treated everolimus-resistant tumours (60 days). Within the everolimus-sensitive group, the combination-treated mice showed the longest median survival (52 days). Of all groups, the everolimus-resistant combination-treated group survived longest (69 days). Combination treatment with everolimus and alpelisib seems promising to overcome everolimus resistance in neuroendocrine neoplasms, and should be further examined in a clinical trial.

The biochemical phenotype of paragangliomas (PGLs) is highly dependent on the underlying genetic background and tumor location. PGLs at extra-adrenal locations usually do not express phenylethanolamine N-methyltransferase (PNMT), the enzyme required for epinephrine production, which was explained by the absence of glucocorticoids. PGLs with pathogenic variants (PVs) in Harvey rat sarcoma viral oncogene homolog (HRAS) can occur in or outside of the adrenal, but always synthesize epinephrine independently of the localization. Here, we characterize the signaling pathways through which PVs in HRAS influence PNMT expression. Catecholamines, cortisol, and transcriptional features of PGL tissues with known genetic background were analyzed. Genetically modified rat pheochromocytoma cells carrying PVs in Hras were generated and analyzed for regulation of Pnmt expression. Elevated epinephrine contents in PGLs with PVs in HRAS were accompanied by enrichment in mitogen-activated protein kinase (MAPK) signaling compared to PGLs with PVs in genes that activate hypoxia pathways. In vitro, Hras PVs increased Pnmt expression and epinephrine biosynthesis through increased phosphorylation of stimulatory protein 1 via MAPK signaling. Here, we provide a molecular mechanism that explains the PV-dependent epinephrine production of PGLs.

Aggressive pheochromocytomas and paragangliomas (PPGLs) are difficult to treat, and molecular targeting is being increasingly considered, but with variable results. This study investigates established and novel molecular-targeted drugs and chemotherapeutic agents for the treatment of PPGLs in human primary cultures and murine cell line spheroids. In PPGLs from 33 patients, including 7 metastatic PPGLs, we identified germline or somatic driver mutations in 79% of cases, allowing us to assess potential differences in drug responsivity between pseudohypoxia-associated cluster 1-related (n = 10) and kinase signaling-associated cluster 2-related (n = 14) PPGL primary cultures. Single anti-cancer drugs were either more effective in cluster 1 (cabozantinib, selpercatinib, and 5-FU) or similarly effective in both clusters (everolimus, sunitinib, alpelisib, trametinib, niraparib, entinostat, gemcitabine, AR-A014418, and high-dose zoledronic acid). High-dose estrogen and low-dose zoledronic acid were the only single substances more effective in cluster 2. Neither cluster 1- nor cluster 2-related patient primary cultures responded to HIF-2a inhibitors, temozolomide, dabrafenib, or octreotide. We showed particular efficacy of targeted combination treatments (cabozantinib/everolimus, alpelisib/everolimus, alpelisib/trametinib) in both clusters, with higher efficacy of some targeted combinations in cluster 2 and overall synergistic effects (cabozantinib/everolimus, alpelisib/trametinib) or synergistic effects in cluster 2 (alpelisib/everolimus). Cabozantinib/everolimus combination therapy, gemcitabine, and high-dose zoledronic acid appear to be promising treatment options with particularly high efficacy in SDHB-mutant and metastatic tumors. In conclusion, only minor differences regarding drug responsivity were found between cluster 1 and cluster 2: some single anti-cancer drugs were more effective in cluster 1 and some targeted combination treatments were more effective in cluster 2.

Head and neck paragangliomas (HNPGLs) are tumors of parasympathetic origin that occur at variable locations and are often secondary to germline mutations in succinate dehydrogenase (SDH) subunit genes. Occasionally, these tumors produce catecholamines. Here, we assessed whether different locations of HNPGLs relate to the presence of SDHx mutations, catecholamine production and other presentations. In this multicenter study, we collected clinical and biochemical data from 244 patients with HNPGLs and 71 patients without HNPGLs. We clarified that jugulotympanic HNPGLs have distinct features. In particular, 88% of jugulotympanic HNPGLs arose in women, among whom only 24% occurred due to SDHx mutations compared to 55% in men. Jugulotympanic HNPGLs were also rarely bilateral, were of a smaller size and were less often metastatic compared to carotid body and vagal HNPGLs. Furthermore, we showed that plasma concentrations of methoxytyramine (MTY) were higher (P < 0.0001) in patients with HNPGL than without HNPGL, whereas plasma normetanephrine did not differ. Only 3.7% of patients showed strong increases in plasma normetanephrine. Plasma MTY was positively related to tumor size but did not relate to the presence of SDHx mutations or tumor location. Our findings confirm that increases in plasma MTY represent the main catecholamine-related biochemical feature of patients with HNPGLs. We expect that more sensitive analytical methods will make biochemical testing of HNPGLs more practical in the future and enable more than the current 30% of patients to be identified with dopamine-producing HNPGLs. The sex-dependent differences in the development of HNPGLs may have relevance to the diagnosis, management and outcomes of these tumors.

Mutations that drive the stabilization of hypoxia inducible factor 2α (HIF2α) and downstream pseudohypoxic signaling are known to predispose to the development of pheochromocytomas and paragangliomas (PPGLs). However, any role of HIF2α in predisposition to metastatic disease remains unclear. To assess such a role we combined gene-manipulations in pheochromocytoma cell lines with retrospective analyses of patient data and gene expression profiling in tumor specimens. Among 425 patients with PPGLs identified with mutations in tumor-susceptibility genes, those with tumors due to activation of pseudohypoxic pathways had a higher frequency of metastatic disease than those with tumors due to activation of kinase-signaling pathways, even without inclusion of patients with mutations in SDHB (18.6% vs 4.3% in, P < 0.0001). Three out of nine (33%) patients with gain-of-function mutations in HIF2α had metastatic disease. In cell line studies, elevated expression of HIF2α enhanced cell proliferation and led to increased migration and invasion capacity. Moreover, HIF2α expression in HIF2α-deficient cells resulted in increased cell motility, diffuse cluster formation and emergence of pseudopodia indicating changes in cell adhesion and cytoskeletal remodeling. In a mouse liver metastasis model, Hif2a enhanced the metastatic load. Transcriptomics data revealed alterations in focal adhesion and extracellular matrix-receptor interactions in HIF2α-mutated PPGLs. Our translational findings demonstrate that HIF2α supports pro-metastatic behavior in PPGLs, though other factors remain critical for subsequent transition to metastasis. We identified LAMB1 and COL4A2 as new potential therapeutic targets for HIF2α-driven PPGLs. Identified HIF2α downstream targets might open a new therapeutic window for aggressive HIF2α-expressing tumors.

Pancreatic neuroendocrine tumors (panNETs) are often inoperable at diagnosis. The mTORC1 inhibitor everolimus has been approved for the treatment of advanced NETs. However, the regular development of resistance to everolimus limits its clinical efficacy. We established two independent everolimus-resistant panNET (BON1) cell lines (BON1 RR1, BON1 RR2) to find potential mechanisms of resistance. After 24 weeks of permanent exposure to 10 nM everolimus, BON1 RR1 and BON1 RR2 showed stable resistance with cellular survival rates of 96.70% (IC 50  = 5200 nM) and 92.30% (IC 50  = 2500 nM), respectively. The control cell line showed sensitivity to 10 nM everolimus with cellular survival declining to 54.70% (IC 50  = 34 nM). Both resistant cell lines did not regain sensitivity over time and showed persistent stable resistance after a drug holiday of 13 weeks. The mechanisms of resistance in our cell line model included morphological adaptations, G1 cell cycle arrest associated with reduced CDK1(cdc2) expression and decreased autophagy. Cellular migration potential was increased and indirectly linked to c-Met activation. GSK3 was over-activated in association with reduced baseline IRS-1 protein levels. Specific GSK3 inhibition strongly decreased BON1 RR1/RR2 cell survival. The combination of everolimus with the PI3Kα inhibitor BYL719 re-established everolimus sensitivity through GSK3 inhibition and restoration of autophagy. We suggest that GSK3 over-activation combined with decreased baseline IRS-1 protein levels and decreased autophagy may be a crucial feature of everolimus resistance, and hence, a possible therapeutic target., (© 2018 Society for Endocrinology.)

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., (© 2018 Society for Endocrinology.)

Tumourigenesis is a relatively common event in endocrine tissues. Currently, specific guidelines have been developed for common malignant endocrine tumours, which also incorporate advances in molecular targeted therapies (MTT), as in thyroid cancer and in gastrointestinal neuroendocrine malignancies. However, there is little information regarding the role and efficacy of MTT in the relatively rare malignant endocrine tumours mainly involving the adrenal medulla, adrenal cortex, pituitary, and parathyroid glands. Due to the rarity of these tumours and the lack of prospective studies, current guidelines are mostly based on retrospective data derived from surgical, locoregional and ablative therapies, and studies with systemic chemotherapy. In addition, in many of these malignancies the prognosis remains poor with individual patients responding differently to currently available treatments, necessitating the development of new personalised therapeutic strategies. Recently, major advances in the molecular understanding of endocrine tumours based on genomic, epigenomic, and transcriptome analysis have emerged, resulting in new insights into their pathogenesis and molecular pathology. This in turn has led to the use of novel MTTs in increasing numbers of patients. In this review, we aim to present currently existing and evolving data using MTT in the treatment of adrenal, pituitary and malignant parathyroid tumours, and explore the current utility and effectiveness of such therapies and their future evolution., (© 2017 Society for Endocrinology.)

Currently, there is no completely effective therapy available for metastatic phaeochromocytomas (PCCs) and paragangliomas. In this study, we explore new molecular targeted therapies for these tumours, using one more benign (mouse phaeochromocytoma cell (MPC)) and one more malignant (mouse tumour tissue (MTT)) mouse PCC cell line - both generated from heterozygous neurofibromin 1 knockout mice. Several PCC-promoting gene mutations have been associated with aberrant activation of PI3K/AKT, mTORC1 and RAS/RAF/ERK signalling. We therefore investigated different agents that interfere specifically with these pathways, including antagonism of the IGF1 receptor by NVP-AEW541. We found that NVP-AEW541 significantly reduced MPC and MTT cell viability at relatively high doses but led to a compensatory up-regulation of ERK and mTORC1 signalling at suboptimal doses while PI3K/AKT inhibition remained stable. We subsequently investigated the effect of the dual PI3K/mTORC1/2 inhibitor NVP-BEZ235, which led to a significant decrease of MPC and MTT cell viability at doses below 50 nM but again increased ERK signalling. Accordingly, we next examined the combination of NVP-BEZ235 with the established agent lovastatin, as this has been described to inhibit ERK signalling. Lovastatin alone significantly reduced MPC and MTT cell viability at therapeutically relevant doses and inhibited both ERK and AKT signalling, but increased mTORC1/p70S6K signalling. Combination treatment with NVP-BEZ235 and lovastatin showed a significant additive effect in MPC and MTT cells and resulted in inhibition of both AKT and mTORC1/p70S6K signalling without ERK up-regulation. Simultaneous inhibition of PI3K/AKT, mTORC1/2 and ERK signalling suggests a novel therapeutic approach for malignant PCCs.