Opposing effects of cannabidiol in patient-derived neuroendocrine tumor, pheochromocytoma/paraganglioma primary cultures.

CONTEXT: Treatment options for advanced neuroendocrine tumors (NETs), pheochromocytomas and paragangliomas (together PPGLs) are still limited. In recent years, anti-tumor effects of cannabinoids have been reported; however, there are only very limited data available in NETs or PPGLs. OBJECTIVE: Investigation of the effects of cannabidiol (CBD) on patient-derived human NET/PPGL primary cultures and on NET/PPGL cell lines. METHODS: We established primary cultures derived from 46 different patients with PPGLs (n = 35) or NETs (n = 11) who underwent tumor resection at two centers. Treatment of patient primary cultures with clinically relevant doses (5 µM) and slightly higher doses (10 µM) of CBD was performed. RESULTS: We found opposing effects of 5 µM CBD: significant anti-tumor effects in 5/35 (14%) and significant tumor-promoting effects in 6/35 (17%) of PPGL primary cultures. In terms of anti-tumor effects, cluster 2-related PPGLs showed significantly stronger responsivity to CBD compared to cluster 1-related PPGLs (p = 0.042). Of the cluster 2-related tumors, NF1 PPGLs showed strongest responsivity (4/5 PPGL primary cultures with a significant decrease in cell viability were NF1-mutated). We also found opposing effects of 10 µM CBD in PPGLs and NETs: significant anti-tumor effects in 9/33 of PPGL (27%) and 3/11 of NET (27%) primary cultures, significant tumor-promoting effects in 6/33 of PPGL (18%) and 2/11 of NET (18%) primary cultures. CONCLUSIONS: We suggest a potential novel treatment option for some NETs/PPGLs, but also provide evidence for caution when applying cannabinoids as supportive therapy for pain or appetite management to cancer patients, and possibly as health supplements.

Animal and Cell Culture Models of PPGLs - Achievements and Limitations.

Research on rare tumors heavily relies on suitable models for basic and translational research. Paragangliomas (PPGL) are rare neuroendocrine tumors (NET), developing from adrenal (pheochromocytoma, PCC) or extra-adrenal (PGL) chromaffin cells, with an annual incidence of 2-8 cases per million. While most PPGL cases exhibit slow growth and are primarily treated with surgery, limited systemic treatment options are available for unresectable or metastatic tumors. Scarcity of appropriate models has hindered PPGL research, preventing the translation of omics knowledge into drug and therapy development. Human PPGL cell lines are not available, and few animal models accurately replicate the disease's genetic and phenotypic characteristics. This review provides an overview of laboratory models for PPGLs, spanning cellular, tissue, organ, and organism levels. We discuss their features, advantages, and potential contributions to diagnostics and therapeutics. Interestingly, it appears that in the PPGL field, disease models already successfully implemented in other cancers have not been fully explored.

Responses to systemic therapy in metastatic pheochromocytoma/paraganglioma: a retrospective multicenter cohort study.

OBJECTIVE: The therapeutic options for metastatic pheochromocytomas/paragangliomas (mPPGLs) include chemotherapy with cyclophosphamide/vincristine/dacarbazine (CVD), temozolomide monotherapy, radionuclide therapies, and tyrosine kinase inhibitors such as sunitinib. The objective of this multicenter retrospective study was to evaluate and compare the responses of mPPGLs including those with pathogenic variants in succinate dehydrogenase subunit B (SDHB), to different systemic treatments. DESIGN: This is a retrospective analysis of treatment responses of mPPGL patients (n = 74) to systemic therapies. METHODS: Patients with mPPGLs treated at 6 specialized national centers were selected based on participation in the ENSAT registry. Survival until detected progression (SDP) and disease-control rates (DCRs) at 3 months were evaluated based on imaging reports. RESULTS: For the group of patients with progressive disease at baseline (83.8% of 74 patients), the DCR with first-line CVD chemotherapy was 75.0% (n = 4, SDP 11 months; SDHB [n = 1]: DCR 100%, SDP 30 months), with somatostatin peptide receptor-based radionuclide therapy (PPRT) 85.7% (n = 21, SDP 17 months; SDHB [n = 10]: DCR 100%, SDP 14 months), with 131I-meta-iodobenzylguanidine (131I-MIBG) 82.6% (n = 23, SDP 43 months; SDHB [n = 4]: DCR 100%, SDP 24 months), with sunitinib 100% (n = 7, SDP 18 months; SDHB [n = 3]: DCR 100%, SDP 18 months), and with somatostatin analogs 100% (n = 4, SDP not reached). The DCR with temozolomide as second-line therapy was 60.0% (n = 5, SDP 10 months; SDHB [n = 4]: DCR 75%, SDP 10 months). CONCLUSIONS: We demonstrate in a real-life clinical setting that all current therapies show reasonable efficacy in preventing disease progression, and this is equally true for patients with germline SDHB mutations.

Multiple endocrine neoplasia type 4 (MEN4): a thorough update on the latest and least known men syndrome.

PURPOSE: Multiple endocrine neoplasia type 4 (MEN4) is a rare multiglandular endocrine neoplasia syndrome, associated with a wide tumor spectrum but hallmarked by primary hyperparathyroidism, which represents the most common clinical feature, followed by pituitary (functional and non-functional) adenomas, and neuroendocrine tumors. MEN4 clinically overlaps MEN type 1 (MEN1) but differs from it for milder clinical features and an older patient's age at onset. The underlying mutated gene, CDKN1B, encodes the cell cycle regulator p27, implicated in cellular proliferation, motility and apoptosis. Given the paucity of MEN4 cases described in the literature, possible genotype-phenotype correlations have not been thoroughly assessed, and specific clinical recommendations are lacking. The present review provides an extensive overview of molecular genetics and clinical features of MEN4, with the aim of contributing to delineate peculiar strategies for clinical management, screening and follow-up of the last and least known MEN syndrome. METHODS: A literature search was performed through online databases like MEDLINE and Scopus. CONCLUSIONS: MEN4 is much less common that MEN1, tend to present later in life with a more indolent course, although involving the same primary organs as MEN1. As a consequence, MEN4 patients might need specific diagnostic and therapeutic approaches and a different strategy for screening and follow-up. Further studies are needed to assess the real oncological risk of MEN4 carriers, and to establish a standardized screening protocol. Furthermore, a deeper understanding of molecular genetics of MEN4 is needed in order to explore p27 as a novel therapeutic target.

Simultaneous magnetic resonance imaging of pH, perfusion and renal filtration using hyperpolarized 13C-labelled Z-OMPD.

pH alterations are a hallmark of many pathologies including cancer and kidney disease. Here, we introduce [1,5-13C2]Z-OMPD as a hyperpolarized extracellular pH and perfusion sensor for MRI which allows to generate a multiparametric fingerprint of renal disease status and to detect local tumor acidification. Exceptional long T1 of two minutes at 1 T, high pH sensitivity of up to 1.9 ppm per pH unit and suitability of using the C1-label as internal frequency reference enables pH imaging in vivo of three pH compartments in healthy rat kidneys. Spectrally selective targeting of both 13C-resonances enables simultaneous imaging of perfusion and filtration in 3D and pH in 2D within one minute to quantify renal blood flow, glomerular filtration rates and renal pH in healthy and hydronephrotic kidneys with superior sensitivity compared to clinical routine methods. Imaging multiple biomarkers within a single session renders [1,5-13C2]Z-OMPD a promising new hyperpolarized agent for oncology and nephrology.

Identification of a Novel SSTR3 Full Agonist for the Treatment of Nonfunctioning Pituitary Adenomas.

Somatostatin receptor (SSTR) agonists have been extensively used for treating neuroendocrine tumors. Synthetic therapeutic agonists showing selectivity for SSTR2 (Octreotide) or for SSTR2 and SSTR5 (Pasireotide) have been approved for the treatment of patients with acromegaly and Cushing's syndrome, as their pituitary tumors highly express SSTR2 or SSTR2/SSTR5, respectively. Nonfunctioning pituitary adenomas (NFPAs), which express high levels of SSTR3 and show only modest response to currently available SSTR agonists, are often invasive and cannot be completely resected, and therefore easily recur. The aim of the present study was the evaluation of ITF2984, a somatostatin analog and full SSTR3 agonist, as a new potential treatment for NFPAs. ITF2984 shows a 10-fold improved affinity for SSTR3 compared to Octreotide or Pasireotide. Molecular modeling and NMR studies indicated that the higher affinity for SSTR3 correlates with a higher stability of a distorted ß-I turn in the cyclic peptide backbone. ITF2984 induces receptor internalization and phosphorylation, and triggers G-protein signaling at pharmacologically relevant concentrations. Furthermore, ITF2984 displays antitumor activity that is dependent on SSTR3 expression levels in the MENX (homozygous mutant) NFPA rat model, which closely recapitulates human disease. Therefore, ITF2984 may represent a novel therapeutic option for patients affected by NFPA.

Inceptor correlates with markers of prostate cancer progression and modulates insulin/IGF1 signaling and cancer cell migration.

OBJECTIVE: The insulin/insulin-like growth factor 1 (IGF1) pathway is emerging as a crucial component of prostate cancer progression. Therefore, we investigated the role of the novel insulin/IGF1 signaling modulator inceptor in prostate cancer. METHODS: We analyzed the expression of inceptor in human samples of benign prostate epithelium and prostate cancer. Further, we performed signaling and functional assays using prostate cancer cell lines. RESULTS: We found that inceptor was expressed in human benign and malignant prostate tissue and its expression positively correlated with various genes of interest, including genes involved in androgen signaling. In vitro, total levels of inceptor were increased upon androgen deprivation and correlated with high levels of androgen receptor in the nucleus. Inceptor overexpression was associated with increased cell migration, altered IGF1R trafficking and higher IGF1R activation. CONCLUSIONS: Our in vitro results showed that inceptor expression was associated with androgen status, increased migration, and IGF1R signaling. In human samples, inceptor expression was significantly correlated with markers of prostate cancer progression. Taken together, these data provide a basis for investigation of inceptor in the context of prostate cancer.

Metastatic Pheochromocytoma and Paraganglioma: Somatostatin Receptor 2 Expression, Genetics, and Therapeutic Responses.

CONTEXT: Pheochromocytomas and paragangliomas (PPGLs) with pathogenic mutations in the succinate dehydrogenase subunit B (SDHB) are associated with a high metastatic risk. Somatostatin receptor 2 (SSTR2)-dependent imaging is the most sensitive imaging modality for SDHB-related PPGLs, suggesting that SSTR2 expression is a significant cell surface therapeutic biomarker of such tumors. OBJECTIVE: Exploration of the relationship between SSTR2 immunoreactivity and SDHB immunoreactivity, mutational status, and clinical behavior of PPGLs. Evaluation of SSTR-based therapies in metastatic PPGLs. METHODS: Retrospective analysis of a multicenter cohort of PPGLs at 6 specialized Endocrine Tumor Centers in Germany, The Netherlands, and Switzerland. Patients with PPGLs participating in the ENSAT registry were included. Clinical data were extracted from medical records, and immunohistochemistry (IHC) for SDHB and SSTR2 was performed in patients with available tumor tissue. Immunoreactivity of SSTR2 was investigated using Volante scores. The main outcome measure was the association of SSTR2 IHC positivity with genetic and clinical-pathological features of PPGLs. RESULTS: Of 202 patients with PPGLs, 50% were SSTR2 positive. SSTR2 positivity was significantly associated with SDHB- and SDHx-related PPGLs, with the strongest SSTR2 staining intensity in SDHB-related PPGLs (P = .01). Moreover, SSTR2 expression was significantly associated with metastatic disease independent of SDHB/SDHx mutation status (P < .001). In metastatic PPGLs, the disease control rate with first-line SSTR-based radionuclide therapy was 67% (n = 22, n = 11 SDHx), and with first-line "cold" somatostatin analogs 100% (n = 6, n = 3 SDHx). CONCLUSION: SSTR2 expression was independently associated with SDHB/SDHx mutations and metastatic disease. We confirm a high disease control rate of somatostatin receptor-based therapies in metastatic PPGLs.

Tribbles Genes in Gastric Cancer: A Tumor-Suppressive Role for TRIB2.

Tribbles pseudokinases (TRIB1-3) are important signaling modulators involved in several cancers. However, their function in gastric cancer (GC) remains undefined. GC is still a deadly disease since the lack of sensitive and specific biomarkers for early diagnosis and therapy response prediction negatively affects patients' outcome. The identification of novel molecular players may lead to more effective diagnostic and therapeutic avenues. Therefore, we investigated the role of TRIB genes in gastric tumorigenesis. Data mining of the TCGA dataset revealed that chromosomal instability (CIN) tumors have lower TRIB2 and higher TRIB3 expression versus microsatellite instability (MSI)-high tumors, while TRIB1 levels are similar in both tumor types. Moreover, in CIN tumors, low TRIB2 expression is significantly associated with aggressive stage IV disease. As no studies on TRIB2 in GC are available, we focused on this gene for further in vitro analyses. We checked the effect of TRIB2 overexpression (OE) on MKN45 and NCI-N87 CIN GC cell lines. In MKN45 cells, TRIB2 OE reduced proliferation and colony formation ability and induced G2/M arrest, while it decreased the proliferation and cell motility of NCI-N87 cells. These effects were not mediated by the MAPK pathway. Our results suggest a tumor-suppressive function of TRIB2 in GC with a CIN phenotype.

Combined Targeting of Pathogenetic Mechanisms in Pancreatic Neuroendocrine Tumors Elicits Synergistic Antitumor Effects.

Pancreatic neuroendocrine neoplasms (PanNENs) are the second most common malignancy of the pancreas. Surgery remains the only curative treatment for localized disease. For patients with inoperable advanced or metastatic disease, few targeted therapies are available, but their efficacy is unpredictable and variable. Exploiting prior knowledge on pathogenetic processes involved in PanNEN tumorigenesis, we tested buparlisib (PI3K inhibitor) and ribociclib (CDK4/6 inhibitor), as single agents or in combination, in different preclinical models. First, we used cell lines representative of well-differentiated (INS-1E, NT-3) and poorly differentiated (BON-1) PanNENs. The combination of buparlisib with ribociclib reduced the proliferation of 2D and 3D spheroid cultures more potently than the individual drugs. Buparlisib, but not ribociclib, induced apoptosis. The anti-proliferative activity of the drugs correlated with downstream target inhibition at mRNA and protein levels. We then tested the drugs on primary islet microtissues from a genetic PanNET animal model (Men1-defective mice) and from wild-type mice: the drug combination was effective against the former without altering islet cell physiology. Finally, we treated PanNET patient-derived islet-like 3D tumoroids: the combination of buparlisib with ribociclib was effective in three out of four samples. Combined targeting of PI3K and CDK4/6 is a promising strategy for PanNENs spanning various molecular and histo-pathological features.

Obesity and cancer-extracellular matrix, angiogenesis, and adrenergic signaling as unusual suspects linking the two diseases.

Obesity is an established risk factor for several human cancers. Given the association between excess body weight and cancer, the increasing rates of obesity worldwide are worrisome. A variety of obesity-related factors has been implicated in cancer initiation, progression, and response to therapy. These factors include circulating nutritional factors, hormones, and cytokines, causing hyperinsulinemia, inflammation, and adipose tissue dysfunction. The impact of these conditions on cancer development and progression has been the focus of extensive literature. In this review, we concentrate on processes that can link obesity and cancer, and which provide a novel perspective: extracellular matrix remodeling, angiogenesis, and adrenergic signaling. We describe molecular mechanisms involved in these processes, which represent putative targets for intervention. Liver, pancreas, and breast cancers were chosen as exemplary disease models. In view of the expanding epidemic of obesity, a better understanding of the tumorigenic process in obese individuals might lead to more effective treatments and preventive measures.

Angpt2/Tie2 autostimulatory loop controls tumorigenesis.

Invasive nonfunctioning (NF) pituitary neuroendocrine tumors (PitNETs) are non-resectable neoplasms associated with frequent relapses and significant comorbidities. As the current therapies of NF-PitNETs often fail, new therapeutic targets are needed. The observation that circulating angiopoietin-2 (ANGPT2) is elevated in patients with NF-PitNET and correlates with tumor aggressiveness prompted us to investigate the ANGPT2/TIE2 axis in NF-PitNETs in the GH3 PitNET cell line, primary human NF-PitNET cells, xenografts in zebrafish and mice, and in MENX rats, the only autochthonous NF-PitNET model. We show that PitNET cells express a functional TIE2 receptor and secrete bioactive ANGPT2, which promotes, besides angiogenesis, tumor cell growth in an autocrine and paracrine fashion. ANGPT2 stimulation of TIE2 in tumor cells activates downstream cell proliferation signals, as previously demonstrated in endothelial cells (ECs). Tie2 gene deletion blunts PitNETs growth in xenograft models, and pharmacological inhibition of Angpt2/Tie2 signaling antagonizes PitNETs in primary cell cultures, tumor xenografts in mice, and in MENX rats. Thus, the ANGPT2/TIE2 axis provides an exploitable therapeutic target in NF-PitNETs and possibly in other tumors expressing ANGPT2/TIE2. The ability of tumor cells to coopt angiogenic signals classically viewed as EC-specific expands our view on the microenvironmental cues that are essential for tumor progression.

Personalized drug testing in human pheochromocytoma/paraganglioma primary cultures.

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.

Adipocyte-specific tribbles pseudokinase 1 regulates plasma adiponectin and plasma lipids in mice.

OBJECTIVE: Multiple genome-wide association studies (GWAS) have identified SNPs in the 8q24 locus near TRIB1 that are significantly associated with plasma lipids and other markers of cardiometabolic health, and prior studies have revealed the roles of hepatic and myeloid Trib1 in plasma lipid regulation and atherosclerosis. The same 8q24 SNPs are additionally associated with plasma adiponectin levels in humans, implicating TRIB1 in adipocyte biology. Here, we hypothesize that TRIB1 in adipose tissue regulates plasma adiponectin, lipids, and metabolic health. METHODS: We investigate the metabolic phenotype of adipocyte-specific Trib1 knockout mice (Trib1_ASKO) fed on chow and high-fat diet (HFD). Through secretomics of adipose tissue explants and RNA-seq of adipocytes and livers from these mice, we further investigate the mechanism of TRIB1 in adipose tissue. RESULTS: Trib1_ASKO mice have an improved metabolic phenotype with increased plasma adiponectin levels, improved glucose tolerance, and decreased plasma lipids. Trib1_ASKO adipocytes have increased adiponectin production and secretion independent of the known TRIB1 function of regulating proteasomal degradation. RNA-seq analysis of adipocytes and livers from Trib1_ASKO mice indicates that alterations in adipocyte function underlie the observed plasma lipid changes. Adipose tissue explant secretomics further reveals that Trib1_ASKO adipose tissue has decreased ANGPTL4 production, and we demonstrate an accompanying increase in the lipoprotein lipase (LPL) activity that likely underlies the triglyceride phenotype. CONCLUSIONS: This study shows that adipocyte Trib1 regulates multiple aspects of metabolic health, confirming previously observed genetic associations in humans and shedding light on the further mechanisms by which TRIB1 regulates plasma lipids and metabolic health.

Imaging pheochromocytoma in small animals: preclinical models to improve diagnosis and treatment.

Pheochromocytomas (PCCs) and paragangliomas (PGLs), together referred to as PPGLs, are rare chromaffin cell-derived tumors. They require timely diagnosis as this is the only way to achieve a cure through surgery and because of the potentially serious cardiovascular complications and sometimes life-threatening comorbidities that can occur if left untreated. The biochemical diagnosis of PPGLs has improved over the last decades, and the knowledge of the underlying genetics has dramatically increased. In addition to conventional anatomical imaging by CT and MRI for PPGL detection, new functional imaging modalities have emerged as very useful for patient surveillance and stratification for therapy. The availability of validated and predictive animal models of cancer is essential for translating molecular, imaging and therapy response findings from the bench to the bedside. This is especially true for rare tumors, such as PPGLs, for which access to large cohorts of patients is limited. There are few animal models of PPGLs that have been instrumental in refining imaging modalities for early tumor detection, as well as in identifying and evaluating novel imaging tracers holding promise for the detection and/or treatment of human PPGLs. The in vivo PPGL models mainly include xenografts/allografts generated by engrafting rat or mouse cell lines, as no representative human cell line is available. In addition, there is a model of endogenous PCCs (i.e., MENX rats) that was characterized in our laboratory. In this review, we will summarize the contribution that various representative models of PPGL have given to the visualization of these tumors in vivo and we present an example of a tracer first evaluated in MENX rats, and then translated to the detection of these tumors in human patients. In addition, we will illustrate briefly the potential of ex vivo biological imaging of intact adrenal glands in MENX rats.

Gender-Specific Efficacy Revealed by Head-to-Head Comparison of Pasireotide and Octreotide in a Representative In Vivo Model of Nonfunctioning Pituitary Tumors.

Invasive nonfunctioning pituitary tumors (NFPTs) are non-resectable neoplasms associated with frequent relapse and significant comorbidities. Current treatments, including somatostatin receptor 2 (SSTR2)-directed somatostatin analogs (SSAs), often fail against NFPTs. Thus, identifying effective therapies is clinically relevant. As NFPTs express SSTR3 at high levels, pasireotide, a multireceptor-targeted SSA, might be beneficial. Here we evaluated pasireotide in the only representative model of spontaneous NFPTs (MENX rats) in vivo. Octreotide long-acting release (LAR), pasireotide LAR, or placebo, were administered to age-matched, tumor-bearing MENX rats of both sexes for 28 d or 56 d. Longitudinal high-resolution magnetic resonance imaging monitored tumor growth. While tumors in placebo-treated rats increased in volume over time, PTs in drug-treated rats displayed significant growth suppression, and occasional tumor shrinkage. Pasireotide elicited stronger growth inhibition. Radiological responses correlated with tumors' proliferation rates. Both SSAs, but especially pasireotide, were more effective in female vs. male rats. Basal Sstr3 expression was significantly higher in the former group. It is noteworthy that female human NFPTs patients also have a trend towards higher SSTR3 expression. Altogether, our studies provide the rationale for testing pasireotide in patients with residual/recurrent NFPTs. If confirmed, the sex-related SSTR3 expression might be used as criteria to stratify NFPTs patients for treatment with pasireotide.

Mutation of the Cell Cycle Regulator p27kip1 Drives Pseudohypoxic Pheochromocytoma Development.

BACKGROUND: Pseudohypoxic tumors activate pro-oncogenic pathways typically associated with severe hypoxia even when sufficient oxygen is present, leading to highly aggressive tumors. Prime examples are pseudohypoxic pheochromocytomas and paragangliomas (p-PPGLs), neuroendendocrine tumors currently lacking effective therapy. Previous attempts to generate mouse models for p-PPGLs all failed. Here, we describe that the rat MENX line, carrying a Cdkn1b (p27) frameshift-mutation, spontaneously develops pseudohypoxic pheochromocytoma (p-PCC). METHODS: We compared rat p-PCCs with their cognate human tumors at different levels: histology, immunohistochemistry, catecholamine profiling, electron microscopy, transcriptome and metabolome. The vessel architecture and angiogenic potential of pheochromocytomas (PCCs) was analyzed by light-sheet fluorescence microscopy ex vivo and multi-spectral optoacoustic tomography (MSOT) in vivo. RESULTS: The analysis of tissues at various stages, from hyperplasia to advanced grades, allowed us to correlate tumor characteristics with progression. Pathological changes affecting the mitochrondrial ultrastructure where present already in hyperplasias. Rat PCCs secreted high levels of norepinephrine and dopamine. Transcriptomic and metabolomic analysis revealed changes in oxidative phosphorylation that aggravated over time, leading to an accumulation of the oncometabolite 2-hydroxyglutarate, and to hypermethylation, evident by the loss of the epigenetic mark 5-hmC. While rat PCC xenografts showed high oxygenation, induced by massive neoangiogenesis, rat primary PCC transcriptomes possessed a pseudohypoxic signature of high Hif2a, Vegfa, and low Pnmt expression, thereby clustering with human p-PPGL. CONCLUSION: Endogenous rat PCCs recapitulate key phenotypic features of human p-PPGLs. Thus, MENX rats emerge as the best available animal model of these aggressive tumors. Our study provides evidence of a link between cell cycle dysregulation and pseudohypoxia.

miR-34a is upregulated in AIP-mutated somatotropinomas and promotes octreotide resistance.

Pituitary adenomas (PAs) are intracranial tumors associated with significant morbidity due to hormonal dysregulation, mass effects and have a heavy treatment burden. Growth hormone (GH)-secreting PAs (somatotropinomas) cause acromegaly-gigantism. Genetic forms of somatotropinomas due to germline AIP mutations (AIPmut+) have an early onset and are aggressive and resistant to treatment with somatostatin analogs (SSAs), including octreotide. The molecular underpinnings of these clinical features remain unclear. We investigated the role of miRNA dysregulation in AIPmut+ vs AIPmut- PA samples by array analysis. miR-34a and miR-145 were highly expressed in AIPmut+ vs AIPmut- somatotropinomas. Ectopic expression of AIPmut (p.R271W) in Aip-/- mouse embryonic fibroblasts (MEFs) upregulated miR-34a and miR-145, establishing a causal link between AIPmut and miRNA expression. In PA cells (GH3), miR-34a overexpression promoted proliferation, clonogenicity, migration and suppressed apoptosis, whereas miR-145 moderately affected proliferation and apoptosis. Moreover, high miR-34a expression increased intracellular cAMP, a critical mitogenic factor in PAs. Crucially, high miR-34a expression significantly blunted octreotide-mediated GH inhibition and antiproliferative effects. miR-34a directly targets Gnai2 encoding Gαi2, a G protein subunit inhibiting cAMP production. Accordingly, Gαi2 levels were significantly lower in AIPmut+ vs AIPmut- PA. Taken together, somatotropinomas with AIP mutations overexpress miR-34a, which in turn downregulates Gαi2 expression, increases cAMP concentration and ultimately promotes cell growth. Upregulation of miR-34a also impairs the hormonal and antiproliferative response of PA cells to octreotide. Thus, miR-34a is a novel downstream target of mutant AIP that promotes a cellular phenotype mirroring the aggressive clinical features of AIPmut+ acromegaly.