Somatostatin Type Research Articles

SSTR2 Mediates the Inhibitory Effect of SST/CST on Lipolysis in Chicken Adipose Tissue.

Somatostatin shows an anti-lipolytic effect in both chickens and ducks. However, its molecular mediator remains to be identified. Here, we report that somatostatin type 2 receptor (SSTR2) is expressed at a high level in chicken adipose tissue. In cultured chicken adipose tissue, the inhibition of glucagon-stimulated lipolysis by somatostatin was blocked by an SSTR2 antagonist (CYN-154086), supporting an SSTR2-mediated anti-lipolytic effect. Furthermore, a significant pro-proliferative effect was detected in SST28-treated immortalized chicken preadipocytes (ICP-1), and this cell proliferative effect may be mediated through the MAPK/ERK signaling pathway activated by SSTR2. In summary, our results demonstrate that SSTR2 may regulate adipose tissue development by affecting the number and volume of adipocytes in chickens.

J-2156, a small molecule somatostatin type 4 receptor agonist, alleviated hindpaw hypersensitivity in the streptozotocin-induced rat model of painful diabetic neuropathy but with a 2-fold decrease in potency at an advanced stage in the model, mimicking morphine.

There is a large unmet need for novel pain-killers to improve relief of painful diabetic neuropathy (PDN). Herein, we assessed the efficacy of the somatostatin type 4 (SST4) receptor agonist, J-2156, for relief of PDN in rats. Diabetes was induced with streptozotocin (STZ; 70mg/kg) and bilateral hindpaw hypersensitivity was fully developed by 8-week post-STZ. In the intervals, 8-12-weeks (morphine-sensitive phase; Phase 1) and 16-18-weeks (morphine-hyposensitive phase; Phase 2) post-STZ, rats received a single dose of intraperitoneal (i.p.) J-2156 (10, 20, 30mg/kg), gabapentin (100mg/kg i.p.), subcutaneous morphine (1mg/kg) or vehicle. Hindpaw withdrawal thresholds (PWTs) were assessed using von Frey filaments pre-dose and at regular intervals over 3-h post-dose. In Phase 1, J-2156at 30mg/kg evoked significant anti-allodynia in the hindpaws with maximal effect at 1.5h compared with 1h for gabapentin and morphine. The durations of action for all three compounds were greater than 3h. The corresponding mean (±SEM) extent and duration of anti-allodynia (ΔPWT AUC) for gabapentin did not differ significantly from that for J-2156 (30mg/kg) or morphine. However, in Phase 2, the ΔPWT AUC for morphine was reduced to approximately 25% of that in Phase 1, mirroring our previous work. Similarly, the mean (±SEM) ΔPWT AUC for J-2156 (30mg/kg) in Phase 2 was approximately 45% of that for Phase 1 whereas for gabapentin the mean (±SEM) ΔPWT AUCs did not differ significantly (p > 0.05) between the two phases. Our findings further describe the preclinical pain relief profile of J-2156 and complement previous work in rat models of inflammatory pain, neuropathic pain and low back pain. SST4 receptor agonists hold promise as novel therapeutics for the relief of PDN, a type of peripheral neuropathic pain that is often intractable to relief with clinically used drug treatment options.

The heterobivalent (SSTR2/albumin) radioligand [67Cu]Cu-NODAGA-cLAB4-TATE enables efficient somatostatin receptor radionuclide theranostics.

Somatostatin type 2 receptor (SSTR2) radionuclide therapy using β- particle-emitting radioligands has entered clinical practice for the treatment of neuroendocrine neoplasms (NENs). Despite the initial success of [177Lu]Lu‑DOTA-TATE, theranostic SSTR2 radioligands require improved pharmacokinetics and enhanced compatibility with alternative radionuclides. Consequently, this study evaluates the pharmacokinetic effects of the albumin-binding domain cLAB4 on theranostic performance of copper‑67-labeled NODAGA-TATE variants in an SSTR2-positive mouse pheochromocytoma (MPC) model. Methods: Binding, uptake, and release of radioligands as well as growth-inhibiting effects were characterized in cells grown as monolayers and spheroids. Tissue pharmacokinetics, absorbed tumor doses, and projected human organ doses were determined from quantitative SPECT imaging in a subcutaneous tumor allograft mouse model. Treatment effects on tumor growth, leukocyte numbers, and renal albumin excretion were assessed. Results: Both copper‑64- and copper‑67-labeled versions of NODAGA-TATE and NODAGA-cLAB4‑TATE showed similar SSTR2 binding affinity, but faster release from tumor cells compared to the clinical reference [177Lu]Lu‑DOTA-TATE. The bifunctional SSTR2/albumin-binding radioligand [67Cu]Cu‑NODAGA-cLAB4‑TATE showed both an improved uptake and prolonged residence time in tumors resulting in equivalent treatment efficacy to [177Lu]Lu‑DOTA-TATE. Absorbed doses were well tolerated in terms of leukocyte counts and kidney function. Conclusion: This preclinical study demonstrates therapeutic efficacy of [67Cu]Cu‑NODAGA-cLAB4‑TATE in SSTR2-positive tumors. As an intrinsic radionuclide theranostic agent, the radioligand provides stable radiocopper complexes and high sensitivity in SPECT imaging for prospective determination and monitoring of therapeutic doses in vivo. Beyond that, copper‑64- and copper‑61-labeled versions offer possibilities for pre- and post-therapeutic PET. Therefore, NODAGA-cLAB4-TATE has the potential to advance clinical use of radiocopper in SSTR2-targeted cancer theranostics.

Epigenetic drugs in somatostatin type 2 receptor radionuclide theranostics and radiation transcriptomics in mouse pheochromocytoma models.

Pheochromocytomas and paragangliomas (PCCs/PGLs) are catecholamine-producing tumors. In inoperable and metastatic cases, somatostatin type 2 receptor (SSTR2) expression allows for peptide receptor radionuclide therapy with [177Lu]Lu-DOTA-TATE. Insufficient receptor levels, however, limit treatment efficacy. This study evaluates whether the epigenetic drugs valproic acid (VPA) and 5-Aza-2'-deoxycytidine (DAC) modulate SSTR2 levels and sensitivity to [177Lu]Lu-DOTA-TATE in two mouse PCC models (MPC and MTT). Methods: Drug-effects on Sstr2/SSTR2 were investigated in terms of promoter methylation, mRNA and protein levels, and radiotracer binding. Radiotracer uptake was measured in subcutaneous allografts in mice using PET and SPECT imaging. Tumor growth and gene expression (RNAseq) were characterized after drug treatments. Results: DAC alone and in combination with VPA increased SSTR2 levels along with radiotracer uptake in vitro in MPC (high-SSTR2) and MTT cells (low-SSTR2). MTT but not MPC allografts responded to DAC and VPA combination with significantly elevated radiotracer uptake, although activity concentrations remained far below those in MPC tumors. In both models, combination of DAC, VPA and [177Lu]Lu-DOTA-TATE was associated with additive effects on tumor growth delay and specific transcriptional responses in gene sets involved in cancer and treatment resistance. Effects of epigenetic drugs were unrelated to CpG island methylation of the Sstr2 promoter. Conclusion: This study demonstrates that SSTR2 induction in mouse pheochromocytoma models has some therapeutic benefit that occurs via yet unknown mechanisms. Transcriptional changes in tumor allografts associated with epigenetic treatment and [177Lu]Lu-DOTA-TATE provide first insights into genetic responses of PCCs/PGLs, potentially useful for developing additional strategies to prevent tumor recurrence.

Toward Personalized Medicine: One Chelator for Imaging and Therapy with Lutetium-177 and Actinium-225.

We report a nonadentate bispidine (3,7-diazabicyclo[3.3.1]nonane) that unveils the potential to bind theranostically relevant radionuclides, including indium-111, lutetium-177, and actinium-225 under mild labeling conditions. This radiopharmaceutical candidate allows the simultaneous application of imaging and treatment (radionuclide theranostics) without changing the type of the bioconjugate; that is, it allows the strong binding to an imaging and a therapeutic radionuclide by the same chelator. Since sophisticated coordination chemistry is required to achieve high thermodynamic and kinetic stability (inertness), it is not surprising that only a few chelators have been reported that are able to strongly bind several radionuclides to a satisfactory extent. Bispidine-derived ligands have proven to be ideal for di- and trivalent metal ions with generally fast complexation kinetics and high in vitro and in vivo stabilities. The presented (radio)complexes are formed under mild conditions (pH 6, <40 °C) and exhibit thermodynamic stability and inertness in human serum comparable to the corresponding DOTA complexes. The bispidine-based complexing agent was conjugated to a peptide, targeting somatostatin type 2 receptors (SSTR2), overexpressed on neuroendocrine tumors. The 177Lu- and 225Ac-labeled conjugates were investigated, considering their binding to two different SSTR2-positive cell lines, including the human pancreatic carcinoid tumor (BON-SSTR2+) and the murine pheochromocytoma cell line (MPC). The biodistribution and accumulation pattern in MPC tumor-bearing mice was also evaluated. The LuIII and AcIII complexes studied show how ligand structures can be optimized in general by extending the denticity and varying the donor set in order to allow for fast complex formation and medically relevant inertness.

O-12 - Epigenetic modifiers in somatostatin type 2 receptor radionuclide theranostics and early transcriptional response of mouse pheochromocytoma models

O-12 - Epigenetic modifiers in somatostatin type 2 receptor radionuclide theranostics and early transcriptional response of mouse pheochromocytoma models

Somatostatin contributes to long-term potentiation at excitatory synapses onto hippocampal somatostatinergic interneurons

Somatostatin-expressing interneurons (SOM-INs) are a major subpopulation of GABAergic cells in CA1 hippocampus that receive excitation from pyramidal cells (PCs), and, in turn, provide feedback inhibition onto PC dendrites. Excitatory synapses onto SOM-INs show a Hebbian long-term potentiation (LTP) mediated by type 1a metabotropic glutamate receptors (mGluR1a) that is implicated in hippocampus-dependent learning. The neuropeptide somatostatin (SST) is also critical for hippocampal long-term synaptic plasticity, as well as learning and memory. SST effects on hippocampal PCs are well documented, but its actions on inhibitory interneurons remain largely undetermined. In the present work, we investigate the involvement of SST in long-term potentiation of CA1 SOM-IN excitatory synapses using pharmacological approaches targeting the somatostatinergic system and whole cell recordings in slices from transgenic mice expressing eYFP in SOM-INs. We report that application of exogenous SST14 induces long-term potentiation of excitatory postsynaptic potentials in SOM-INs via somatostatin type 1–5 receptors (SST1-5Rs) but does not affect synapses of PC or parvalbumin-expressing interneurons. Hebbian LTP in SOM-INs was prevented by inhibition of SSTRs and by depletion of SST by cysteamine treatment, suggesting a critical role of endogenous SST in LTP. LTP of SOM-IN excitatory synapses induced by SST14 was independent of NMDAR and mGluR1a, activity-dependent, and prevented by blocking GABAA receptor function. Our results indicate that endogenous SST may contribute to Hebbian LTP at excitatory synapses of SOM-INs by controlling GABAA inhibition, uncovering a novel role for SST in regulating long-term synaptic plasticity in somatostatinergic cells that may be important for hippocampus-dependent memory processes.

Safety and Efficacy of Switching Injected Peptide Long-Acting Somatostatin Receptor Ligands to Once Daily Oral Paltusotine: ACROBAT Edge Phase 2 Study

Patients with acromegaly not cured by surgery are often initially treated with injected peptide long-acting somatostatin receptor ligands (SRLs). Non-peptide small molecules can also activate the somatostatin receptor and do so with a high degree of precision for the target therapeutic receptor subtype. Paltusotine (formerly CRN00808) is a small molecule somatostatin type 2 (SST2) receptor agonist with high oral bioavailability (70%) and pharmacokinetic profile suitable for once daily dosing. In healthy volunteers, paltusotine has been shown to lower growth hormone (GH) and insulin-like growth factor-1 (IGF-1) levels. We hypothesized that patients with acromegaly could switch from injected SRLs to once daily oral paltusotine while maintaining baseline IGF-1 levels. ACROBAT Edge (NCT03789656) was a single-arm study designed to evaluate the safety and efficacy of switching from injected SRLs to paltusotine in patients with acromegaly. The primary analysis population consisted of those who had not achieved normal IGF-1 levels despite stable therapy with long-acting octreotide or lanreotide. Eligible patients received their last injection of SRL 4 weeks prior to switching to once daily oral paltusotine monotherapy for a 13-week treatment period. The starting dose of 10 mg per day was uptitrated in 10 mg increments at specified study visits to a maximal dose of 40 mg per day based on protocol specified study drug toleration and IGF-1 criteria. The primary endpoint was change in IGF-1 from baseline to the completion of the 13-week treatment period. Statistical testing was based on non-parametric Wilcoxon Sign Rank test of whether the median change is different from zero. In addition, the rise in IGF-1 during a 4-week washout period was used to provide supportive evidence of efficacy. Twenty-five patients were enrolled in the primary analysis group, three patients discontinued from the study for non-study drug related reasons, two during the treatment period and one during the washout period after completing treatment. The primary endpoint was achieved as paltusotine treatment resulted in no significant change in IGF-1 levels at week 13 compared to baseline [change in IGF-1 =-0.034 (-0.107, 0.107), median (IQR), p>0.6]. Of the 23 patients who completed the dosing period, 20 (87%) achieved IGF-1 levels at the end of treatment that were within 20% of baseline or lower. Median IGF-1 values rose significantly after paltusotine washout (p<0.0001). The most common treatment-emergent adverse events (>10%) included: headache, arthralgia, fatigue, peripheral swelling, paresthesia and hyperhidrosis. There were no discontinuations due to adverse events and no treatment related serious adverse events. These results suggest that patients with acromegaly treated with injected SRLs can switch to oral paltusotine while maintaining IGF-1 and that paltusotine appeared to be well tolerated.

HIF2alpha-Associated Pseudohypoxia Promotes Radioresistance in Pheochromocytoma: Insights from 3D Models

Simple SummaryLow oxygen levels (hypoxia) as well as genetic defects activating hypoxia signaling pathways (pseudohypoxia) are known to contribute to tumorigenesis and therapy resistance in various cancers. The genetic background of pheochromocytomas and paragangliomas is well characterized and indicates that pseudohypoxia plays a role in tumor formation and metastatic spread in a subgroup of these tumors. It is, however, unknown how pseudohypoxia affects susceptibility to radiation treatments, which is of particular interest, since targeted radionuclide therapy is one of the few options used against metastatic pheochromocytomas and paragangliomas. To date, no curative treatment is available for metastatic disease. Here, we report on the radioprotective effects of pseudohypoxia against both external irradiation and beta particle-emitting lutetium-177 in a pheochromocytoma tumor spheroid model expressing hypoxia-inducible factor 2 alpha. Our findings highlight hypoxia signaling pathways as potential targets for neo-adjuvant—in particular, radiosensitizing—therapies in pseudohypoxic pheochromocytomas and paragangliomas.Pheochromocytomas and paragangliomas (PCCs/PGLs) are rare neuroendocrine tumors arising from chromaffin tissue located in the adrenal or ganglia of the sympathetic or parasympathetic nervous system. The treatment of non-resectable or metastatic PCCs/PGLs is still limited to palliative measures, including somatostatin type 2 receptor radionuclide therapy with [177Lu]Lu-DOTA-TATE as one of the most effective approaches to date. Nevertheless, the metabolic and molecular determinants of radiation response in PCCs/PGLs have not yet been characterized. This study investigates the effects of hypoxia-inducible factor 2 alpha (HIF2α) on the susceptibility of PCCs/PGLs to radiation treatments using spheroids grown from genetically engineered mouse pheochromocytoma (MPC) cells. The expression of Hif2α was associated with the significantly increased resistance of MPC spheroids to external X-ray irradiation and exposure to beta particle-emitting [177Lu]LuCl3 compared to Hif2α-deficient controls. Exposure to [177Lu]LuCl3 provided an increased long-term control of MPC spheroids compared to single-dose external X-ray irradiation. This study provides the first experimental evidence that HIF2α-associated pseudohypoxia contributes to a radioresistant phenotype of PCCs/PGLs. Furthermore, the external irradiation and [177Lu]LuCl3 exposure of MPC spheroids provide surrogate models for radiation treatments to further investigate the metabolic and molecular determinants of radiation responses in PCCs/PGLs and evaluate the effects of neo-adjuvant—in particular, radiosensitizing—treatments in combination with targeted radionuclide therapies.

Comparison of Antinociceptive Effect of Octreotide With Morphine in a Rat Model of Acute Inflammatory Pain

Background:Opioids such as morphine are used for treating moderate to severe pain. However, they also produce adverse effects such as nausea, constipation, addiction, and respiratory depression. Thus, other suitable analgesics need to be identified. Somatostatin is an inhibitory neuropeptide that modulates the transmission of pain. However, the half-life of somatostatin is short. In the present study, the antinociceptive effect of octreotide (a stable long-acting analog of somatostatin) was evaluated in rats with acute inflammatory pain.Methods:Sprague Dawley rats (n = 42) were divided into control (n = 6) and carrageenan injected groups (n = 36). The carrageena group was divided into three equal subgroups and treated with saline, morphine (10 mg/kg), and octreotide (3 µg). Rats belonging to each subgroup (n = 12) were again randomly divided into two equal sets. They were subjected to (a) behavioral evaluation of pain (allodynia) and estimation of paw edema, followed by immunohistochemical analysis of the expression of somatostatin type 2 receptor (sst2r) in the spinal cord and (b) estimation of open-field activity. Allodynia and paw edema were measured by von Frey filaments and plethysmometer, respectively, at 3 and 4 h after carrageenan injection. Expression of sst2r was examined after 24 hours, whereas open-field activity was evaluated after 3 hours.Results:In comparison to the saline-treated group, allodynia was partially attenuated by octreotide, though this was almost completely reversed by morphine. Paw edema was unaffected by octreotide, though it was marginally increased by morphine. This was not related to increased activity of rats, following relief from pain. Immunohistochemistry revealed a significant increase in the expression of sst2r in saline-treated rats, but a decrease in other groups.Conclusion:Octreotide has an antinociceptive effect, which was less than morphine. Increased edema following morphine could result from venodilation. Variations in the sst2r expression suggest its involvement in pain modulation at the spinal level. This information may have clinical relevance.

Assessment of the anti-hyperalgesic efficacy of J-2156, relative to clinically available analgesic/adjuvant agents in a rat model of mild to moderate chronic mechanical low back pain (LBP).

Chronic mechanical low back pain (cLBP) is a leading cause of disability and a major socio-economic burden internationally. The lifetime prevalence of non-specific LBP is approximately 84%, with the prevalence of cLBP at about 23%. Clinically available analgesic/adjuvant medications often provide inadequate pain relief in patients experiencing cLBP. Hence, the urgency for discovery of effective and better tolerated medications. Fourteen days after the induction of five shallow annular punctures (5X) in the lumbar intervertebral discs at L4/L5 and L5/L6 in male Sprague-Dawley rats, mechanical hyperalgesia was fully developed in the lumbar axial deep tissues at L4/L5 (primary) and L1 (secondary). Importantly, mechanical allodynia in the hindpaws was absent. From day 28, we assessed the face validity of our mild to moderate LBP-5X rat model using four clinically available analgesic/adjuvant drugs, namely gabapentin, morphine, meloxicam and amitriptyline relative to vehicle. Additionally, the anti-hyperalgesic effects of J-2156, a highly selective small molecule somatostatin type 4 receptor agonist was assessed. Single i.p. bolus doses of gabapentin and meloxicam at the highest doses tested (100 and 30mg/kg, respectively) alleviated secondary hyperalgesia (L1) but not primary hyperalgesia (L4/5). Morphine at 1mg/kg alleviated both primary and secondary hyperalgesia in these tissues, whereas amitriptyline at the doses tested, lacked efficacy. These findings attest to the face validity of our model. J-2156 at 10 and 30mg/kg alleviated secondary hyperalgesia in the lumbar axial deep tissues at L1 with a non-significant trend for relief of primary hyperalgesia in the corresponding tissues at L4/L5 in these animals.

Somatostatin Type 2 Receptor Antibody Enhances Mechanical Hyperalgesia in the Dorsal Root Ganglion Neurons after Sciatic Nerve-pinch Injury: Evidence of Behavioral Studies and Bax Protein Expression.

Our previous study has indicated that somatostatin potently inhibits neuropathic pain through the activation of its type 2 receptor (SSTR2) in mouse dorsal root ganglion and spinal cord. However, the underlying mechanism of this activation has not been elucidated clearly. The aim of this study is to perform the pharmacological studies on the basis of sciatic nerve-pinch mice model and explore the underlying mechanism involving SSTR2. On the basis of a sciatic nerve-pinch injury model, we aimed at comparing the painful behavior and dorsal root ganglion neurons neurochemical changes after the SSTR2 antibody (anti- SSTR2;5μl,1μg/ml) administration in the mouse. After pinch nerve injury, we found that the mechanical hyperalgesia and severely painful behavior (autotomy) were detected after the application of SSTR2 antibody (anti-SSTR2; 5μl, 1μg/ml) on the pinch-injured nerve. The up-regulated phosphorylated ERK (p-ERK) expression and the apoptotic marker (i.e., Bax) were significantly decreased in DRGs after anti-SSTR2 treatment. The current data suggested that inhibitory changes in proteins from the apoptotic pathway in anti-SSTR2-treated groups might be taking place to overcome the protein deficits caused by SSTR2 antibody and supported the new therapeutic intervention with SSTR2 antagonist for neuronal degeneration following nerve injury.

Fluorescent mouse pheochromocytoma spheroids expressing hypoxia-inducible factor 2 alpha: Morphologic and radiopharmacologic characterization

BACKGROUND:Pheochromocytomas and paragangliomas (PPGLs) are rare catecholamine-producing tumors arising from chromaffin tissue. In a PPGL subgroup, dysregulation of hypoxia signaling pathways, in particular mediated through stabilization of hypoxia-inducible factor 2 alpha (HIF2α), have been sugges ted to drive tumorigenesis through altering downstream transcriptional activity. OBJECTIVE:This study evaluated the use of mCherry-transgenic mouse pheochromocytoma (MPCmCherry) spheroids as in vitro models for investigating consequences of HIF2α expression on aggregation behavior, morphology, growth, glucose consumption, amino acid uptake, and somatostatin type 2 receptors under stable hypoxic conditions. METHODS:MPCmCherry spheroids were monitored using confocal laser scanning microscopy. Hypoxic regions were detected using pimonidazole. Radiotracer incubation was performed using 2-[18F]fluoro-2-deoxyglucose ([18F]FDG), O-3-(2[18F]fluoroethoxy)-4-hydroxyphenylalanine ([18F]OFED), and [68Ga]Ga-(Tyr3)octreotate ([68Ga]Ga-DOTA-TATE). RESULTS:Both HIF2α-expressing and empty vector (EV) control spheroids showed regions of stable cellular hypoxia. Expression of HIF2α in MPCmCherry spheroids was associated with less symmetric morphology, faster growth, and decreased uptake of [68Ga]Ga-DOTA-TATE (somatostatin type 2 receptors) compared to controls, whereas, uptake of [18F]FDG (glucose transporter 1 and hexokinases) and [18F]OFED (system L amino acid transporter 1) remained unaffected. CONCLUSIONS:The recent study proved MPCmCherry spheroids to be complex three-dimensional tumor cell models for investigating morphologic and metabolic consequences of dysregulated hypoxia pathways under hypoxic conditions.

Emerging Role of 177Lu in Nuclear Oncology: A Brief Review

With the innovations in nuclear medicine techniques, Lutetium 177 (177Lu) has epitomized as a revolutionary theranostic agent- with both scintigraphic and therapeutic properties. The present review focusses on the introduction of 177Lu as a promising modality for tumor diagnosis and therapy in widespread metastases . Being a shorter β-range emitter providing better irradiation of smaller tumor volumes, 177Lu- based PRRT is being increasingly used in patients with somatostatin receptor positive neuroendocrine tumors. Clinical trials with 177Lu–DOTATATE and 177Lu–DOTATOC have gained considerable interest in recent years with successful tumor regression in patients with malignant metastatic neuroendocrine tumors. Especially, therapy with 177Lu-DOTATATE PRRT has reported to significantly improve the quality of life of Gastroenteropancreatic NET patients because of higher affinity of DOTATATE for the somatostatin type 2 receptors. In addition, this review also sheds light on the diagnostic and palliative aspects of 177Lu which also serves to be an attractive candidate for the preparation of radiopharmaceuticals for radiation synovectomy of small to medium sized joints. Enlisting all the said features, 177Lu is strongly emerging as a promising theranostic agent that could possibly endow Nuclear Medicine an edge over other conventional therapies in near future.

Peptide receptor radionuclide therapy: focus on bronchial neuroendocrine tumors.

Well-differentiated bronchial neuroendocrine tumors (B-NETs) are rare. They represent 1-5% of all lung cancers. The incidence of these neoplasms has risen over the past 30years and, especially for advanced or metastatic disease, management is complex and requires a multidisciplinary approach. Treatment with somatostatin analogs (SSAs) is the most important first-line therapy, in particular in well-differentiated NETs with high somatostatin type receptor (SSTR) expression. In these tumors, the role of mammalian target of rapamycin (m-TOR) inhibitors and the potential utility of other target therapies remain unclear while chemotherapy represents the gold standard treatment only for aggressive forms with low SSTR expression. Peptide receptor radionuclide therapy (PRRT) is an emerging treatment modality for advanced NETs. There are many cumulative evidences about the effectiveness and tolerability of this therapeutic approach, especially in gastro-entero-pancreatic (GEP)-NETs. For B-NETs, scientific research is moving more slowly. Here, we performed a review in order to evaluate the efficacy and toxicity of PRRT with a focus on patients with inoperable or metastatic well-differentiated B-NETs.

Epidrug-induced upregulation of functional somatostatin type 2 receptors in human pancreatic neuroendocrine tumor cells.

Somatostatin receptors are a pivotal target for treatment of pancreatic neuroendocrine tumors (pNET), either with somatostatin analogues (SSA) or radiolabeled SSA. The highest affinity target for the most commonly used SSA is the somatostatin receptor type 2 (sst2). An important factor that may complicate treatment efficacy, is the variable number of receptors expressed on pNETs. Gene expression is subject to complex regulation, in which epigenetics has a central role. In this study we explored the possible role of epigenetic modifications in the variations in sst2 expression levels in two human pNET cell lines, BON-1 and QGP-1. We found upregulation of sst2 mRNA after treatment with the epidrugs 5-aza-2’-deoxycytidine (5-aza-dC) and valproic acid (VPA), an increased uptake of radiolabeled octreotide, as well as increased sensitivity to the SSA octreotide in functional cAMP inhibition. At epigenetic level we observed low methylation levels of the sst2 gene promoter region irrespective of expression. Activating histone mark H3K9Ac can be regulated with epidrug treatment, with an angle of effect corresponding to the effect on mRNA expression. Repressive histone mark H3K27me3 is not regulated by either 5-aza-dC or VPA. We conclude that epidrug treatment, in particular with combined 5-aza-dC and VPA treatment, might hold promise for improving and adding to current SSA treatment strategies of patients with pNETs.

Multimodal Somatostatin Receptor Theranostics Using [(64)Cu]Cu-/[(177)Lu]Lu-DOTA-(Tyr(3))octreotate and AN-238 in a Mouse Pheochromocytoma Model.

Pheochromocytomas and extra-adrenal paragangliomas (PHEO/PGLs) are rare catecholamine-producing chromaffin cell tumors. For metastatic disease, no effective therapy is available. Overexpression of somatostatin type 2 receptors (SSTR2) in PHEO/PGLs promotes interest in applying therapies using somatostatin analogs linked to radionuclides and/or cytotoxic compounds, such as [177Lu]Lu-DOTA-(Tyr3)octreotate (DOTATATE) and AN-238. Systematic evaluation of such therapies for the treatment of PHEO/PGLs requires sophisticated animal models. In this study, the mouse pheochromocytoma (MPC)-mCherry allograft model showed high tumor densities of murine SSTR2 (mSSTR2) and high tumor uptake of [64Cu]Cu-DOTATATE. Using tumor sections, we assessed mSSTR2-specific binding of DOTATATE, AN-238, and somatostatin-14. Therapeutic studies showed substantial reduction of tumor growth and tumor-related renal monoamine excretion in tumor-bearing mice after treatment with [177Lu]Lu-DOTATATE compared to AN-238 and doxorubicin. Analyses did not show agonist-dependent receptor downregulation after single mSSTR2-targeting therapies. This study demonstrates that the MPC-mCherry model is a uniquely powerful tool for the preclinical evaluation of SSTR2-targeting theranostic applications in vivo. Our findings highlight the therapeutic potential of somatostatin analogs, especially of [177Lu]Lu-DOTATATE, for the treatment of metastatic PHEO/PGLs. Repeated treatment cycles, fractionated combinations of SSTR2-targeting radionuclide and cytotoxic therapies, and other adjuvant compounds addressing additional mechanisms may further enhance therapeutic outcome.

Trans-Modulation of the Somatostatin Type 2A Receptor Trafficking by Insulin-Regulated Aminopeptidase Decreases Limbic Seizures.

The somatostatin type 2A receptor (sst2A) is localized on principal hippocampal neurons and displays anticonvulsant properties. Following agonist exposure, however, this receptor rapidly internalizes and recycles slowly. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. We therefore assessed by in vitro and in vivo experiments whether IRAP could regulate the trafficking of this receptor. We demonstrate that IRAP ligands accelerate sst2A recycling in hippocampal neurons. Because IRAP ligands increase the density of sst2A receptors at the plasma membrane, they also potentiate the effects of this inhibitory receptor on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures.

Does Somatostatin or Gastric Inhibitory Peptide Receptor Expression Correlate with Tumor Grade and Stage in Gut Neuroendocrine Tumors?

Background/Aims: Important characteristics of neuroendocrine neoplasms (NEN) for prognosis and therapeutic decisions are the MIB-1 proliferative index (tumor grade) and tumor stage. Moreover, these tumors express peptide hormone receptors like somatostatin and gastric inhibitory peptide (GIP) receptors which represent important established and potential future targets, respectively, for molecular imaging and radiotherapy. However, the interrelation between tumor proliferation, stage, and peptide receptor amounts has never been assessed. Methods: In 114 gastrointestinal and bronchopulmonary NEN, the proliferative rate assessed with MIB-1 immunohistochemistry and tumor stage were compared with the somatostatin type 2 receptor (sst2) and GIP receptor expression measured quantitatively with in vitro receptor autoradiography. Results: NEN generally showed high sst2 and GIP receptor expression. GIP receptor but not sst2 expression correlated with the MIB-1 index. GIP receptor levels gradually increased in a subset of insulinomas and nonfunctioning pancreatic NEN, and decreased in ileal and bronchopulmonary NEN with increasing MIB-1 rate. MIB-1 levels were identified, above which GIP receptor levels were consistently high or low. These MIB-1 levels were clearly different from those defining tumor grade. In grade 3 NEN, GIP receptor levels were always low, while sst2 levels were variable and sometimes extremely high. Conversely, sst2 expression correlated more frequently with tumor stage than GIP receptor expression, with metastasized NEN showing higher sst2 levels than localized tumors. Conclusions: sst2, a clinically crucial molecular target, shows variable and unpredictable expression in NEN irrespective of tumor grade. Therefore, each NEN should be tested for sst2 if clinical applications with somatostatin analogs are considered. Conversely, the potential future role of GIP receptors as molecular targets in NEN may be dependent on the MIB-1 level.

Activation of ras‐dependent signaling pathways by G14‐coupled receptors requires the adaptor protein TPR1

Many G(q) -coupled receptors mediate mitogenic signals by stimulating extracellular signal-regulated protein kinases (ERKs) that are typically regulated by the small GTPase Ras. Recent studies have revealed that members of the Gα(q) family may possess the ability to activate Ras/ERK by interacting with the adaptor protein tetratricopeptide repeat 1 (TPR1). Within the Gα(q) family, the highly promiscuous Gα(14) can relay signals from numerous receptors. Here, we examined if Gα(14) interacts with TPR1 to stimulate Ras signaling pathways. Expression of the constitutively active Gα(14) QL mutant in HEK293 cells led to the formation of GTP-bound Ras as well as increased phosphorylations of downstream signaling molecules including ERK and IκB kinase. Stimulation of endogenous G(14) -coupled somatostatin type 2 and α(2) -adrenergic receptors produced similar responses in human hepatocellular HepG2 carcinoma cells. Co-immunoprecipitation assays using HEK293 cells demonstrated a stronger association of TPR1 for Gα(14) QL than Gα(14) , suggesting that TPR1 preferentially binds to the GTP-bound form of Gα(14) . Activated Gα(14) also interacted with the Ras guanine nucleotide exchange factors SOS1 and SOS2. Expression of a dominant negative mutant of TPR1 or siRNA-mediated knockdown of TPR1 effectively abolished the ability of Gα(14) to induce Ras signaling in native HepG2 or transfected HEK293 cells. Although expression of the dominant negative mutant of TPR1 suppressed Gα(14) QL-induced phosphorylations of ERK and IκB kinase, it did not affect Gα(14) QL-induced stimulation of phospholipase Cβ or c-Jun N-terminal kinase. Our results suggest that TPR1 is required for Gα(14) to stimulate Ras-dependent signaling pathways, but not for the propagation of signals along Ras-independent pathways.