Targeting pheochromocytoma/paraganglioma with polyamine inhibitors.

BACKGROUND: Pheochromocytomas (PCCs) and paragangliomas (PGLs) are neuroendocrine tumors that are mostly benign. Metastatic disease does occur in about 10% of cases of PCC and up to 25% of PGL, and for these patients no effective therapies are available. Patients with mutations in the succinate dehydrogenase subunit B (SDHB) gene tend to have metastatic disease. We hypothesized that a down-regulation in the active succinate dehydrogenase B subunit should result in notable changes in cellular metabolic profile and could present a vulnerability point for successful pharmacological targeting. METHODS: Metabolomic analysis was performed on human hPheo1 cells and shRNA SDHB knockdown hPheo1 (hPheo1 SDHB KD) cells. Additional analysis of 115 human fresh frozen samples was conducted. In vitro studies using N1,N11-diethylnorspermine (DENSPM) and N1,N12- diethylspermine (DESPM) treatments were carried out. DENSPM efficacy was assessed in human cell line derived mouse xenografts. RESULTS: Components of the polyamine pathway were elevated in hPheo1 SDHB KD cells compared to wild-type cells. A similar observation was noted in SDHx PCC/PGLs tissues compared to their non-mutated counterparts. Specifically, spermidine, and spermine were significantly elevated in SDHx-mutated PCC/PGLs, with a similar trend in hPheo1 SDHB KD cells. Polyamine pathway inhibitors DENSPM and DESPM effectively inhibited growth of hPheo1 cells in vitro as well in mouse xenografts. CONCLUSIONS: This study demonstrates overactive polyamine pathway in PCC/PGL with SDHB mutations. Treatment with polyamine pathway inhibitors significantly inhibited hPheo1 cell growth and led to growth suppression in xenograft mice treated with DENSPM. These studies strongly implicate the polyamine pathway in PCC/PGL pathophysiology and provide new foundation for exploring the role for polyamine analogue inhibitors in treating metastatic PCC/PGL. PRéCIS: Cell line metabolomics on hPheo1 cells and PCC/PGL tumor tissue indicate that the polyamine pathway is activated. Polyamine inhibitors in vitro and in vivo demonstrate that polyamine inhibitors are promising for malignant PCC/PGL treatment. However, further research is warranted.

A xenograft and cell line model of SDH-deficient pheochromocytoma derived from Sdhb+/- rats.

Tumors caused by loss-of-function mutations in genes encoding TCA cycle enzymes have been recently discovered and are now of great interest. Mutations in succinate dehydrogenase (SDH) subunits cause pheochromocytoma/paraganglioma (PCPG) and syndromically associated tumors, which differ phenotypically and clinically from more common SDH-intact tumors of the same types. Consequences of SDH deficiency include rewired metabolism, pseudohypoxic signaling and altered redox balance. PCPG with SDHB mutations are particularly aggressive, and development of treatments has been hampered by lack of valid experimental models. Attempts to develop mouse models have been unsuccessful. Using a new strategy, we developed a xenograft and cell line model of SDH-deficient pheochromocytoma from rats with a heterozygous germline Sdhb mutation. The genome, transcriptome and metabolome of this model, called RS0, closely resemble those of SDHB-mutated human PCPGs, making it the most valid model now available. Strategies employed to develop RS0 may be broadly applicable to other SDH-deficient tumors.

A Previously Unrecognized Monocytic Component of Pheochromocytoma and Paraganglioma.

We describe a consistently present, previously unrecognized, population of monocytes in pheochromocytomas and paragangliomas. Although sustentacular cells are generally recognized as a common component of these tumors, differential immunohistochemical staining for CD163 and S100 shows that monocytes can in fact be more numerous. These cells frequently resemble sustentacular cells topographically and cytologically, possibly explaining why they have not been previously noticed. They contribute to the tumor proteome and may have implications for tumor biology. No correlations were identifiable between the presence of these cells and any clinical characteristics of the tumors in the present study. A possible association with genotype is suggested by immunoblot showing high expression of CD163 protein in tumors with succinate dehydrogenase mutations.

A unique model for SDH-deficient GIST: an endocrine-related cancer.

We describe a unique patient-derived xenograft (PDX) and cell culture model of succinate dehydrogenase-deficient gastrointestinal stromal tumor (SDH-deficient GIST), a rare mesenchymal tumor that can occur in association with paragangliomas in hereditary and non-hereditary syndromes. This model is potentially important for what it might reveal specifically pertinent to this rare tumor type and, more broadly, to other types of SDH-deficient tumors. The primary tumor and xenografts show a very high proliferative fraction, and distinctive morphology characterized by tiny cells with marked autophagic activity. It is likely that these characteristics resulted from the combination of the germline SDHB mutation and a somatic KRAS G12D mutation. The most broadly relevant findings to date concern oxygen and oxidative stress. In paragangliomas harboring SDHx mutations, both hypoxic signaling and oxidative stress are putative drivers of tumor growth. However, there are no models for SDH-deficient paragangliomas. This related model is the first from a SDHB-mutated human tumor that can be experimentally manipulated to study mechanisms of oxygen effects and novel treatment strategies. Our data suggest that tumor growth and survival require a balance between protective effects of hypoxic signaling vs deleterious effects of oxidative stress. While reduced oxygen concentration promotes tumor cell survival, a further survival benefit is achieved with antioxidants. This suggests potential use of drugs that increase oxidative stress as novel therapies. In addition, autophagy, which has not been reported as a major finding in any type of SDH-deficient tumor, is a potential target of agents that might trigger autophagic cell death.

Acute Hypoxia Induces Enkephalin Production and Release in an Adrenergic Cell Line Model of Neonatal Chromaffin Cell Responses to Hypoxic Stress.

OBJECTIVE: Prior to maturation of the human sympathetic nervous system, the neonatal adrenal medulla senses and responds to hypoxia. In addition to catecholamine release, the adrenal medulla synthesizes and stores opioid peptides, notably enkephalin (ENK). However, it is not known whether acute hypoxia evokes adrenal ENK production and release, as seen in the central nervous system (CNS). We hypothesize that acute hypoxia stimulates synthesis and release of ENK in chromaffin cells. STUDY DESIGN: Cultures of adrenergic mouse pheochromocytoma cells (MPC) 10/9/96CR were incubated in 10% oxygen (O2) at intervals of up to 60 minutes. ENK content and release were measured by Met-ENK enzyme-linked immunosorbent assay (ELISA). ENK messenger ribonucleic acid (mRNA) was analyzed by quantitative reverse-transcriptase polymerase chain reaction (PCR). RESULTS: Incubation of MPC 10/9 cells in 10% O2 evoked rapid release of epinephrine and of Met-ENK which increased approximately twofold in 15 minutes. Reduced [O2] also induced an overall increase (14%) in cellular ENK peptide content within 60 minutes. Acute hypoxia-stimulated release of Met-ENK was accompanied by increased mRNAENK expression in MPC 10/9s, a cell culture model of adrenergic chromaffin cells. CONCLUSION: We speculate that the ability of reduced [O2] to evoke ENK release from chromaffin cells may influence blood pressure regulation and heart contractility, thereby providing an adaptive survival advantage during neonatal asphyxia.

Case Report of a Prolactinoma in a Patient With a Novel MAX Mutation and Bilateral Pheochromocytomas.

Pheochromocytomas are neuroendocrine tumors that can arise sporadically or be inherited as a familial disease, and they may occur in isolation or as part of a multitumor syndrome. Familial disease typically presents in younger patients with a higher risk of multifocality. Recently, the tumor suppressor MYC-associated factor X (MAX) gene has been implicated as a cause of familial isolated pheochromocytoma and paraganglioma. We describe a patient with a pituitary prolactinoma and bilateral pheochromocytomas who tested positive for a germline MAX mutation. Interestingly, the patient also had mild primary hyperparathyroidism that resolved upon resection of the pheochromocytomas despite the absence of parathyroid hormone staining in the tumors. To our knowledge, this case is the first report of prolactinoma in a patient with a MAX mutation, which suggests the possibility of germline MAX mutations also contributing to the development of prolactinomas.

Pathology of Human Pheochromocytoma and Paraganglioma Xenografts in NSG Mice.

A major impediment to the development of effective treatments for metastatic or unresectable pheochromocytomas and paragangliomas has been the absence of valid models for pre-clinical testing. Attempts to establish cell lines or xenografts from human pheochromocytomas and paragangliomas have previously been unsuccessful. NOD-scid gamma (NSG) mice are a recently developed strain lacking functional B-cells, T-cells, and NK cells. We report here that xenografts of primary human paragangliomas will take in NSG mice while maintaining their architectural and immunophenotypic characteristics as expressed in the patients. In contrast to grafts of cell lines and of most common types of primary tumors, the growth rate of grafted paragangliomas is very slow, accurately representing the growth rate of most pheochromocytomas and paragangliomas even in metastases in humans. Although the model is therefore technically challenging, primary patient-derived xenografts of paragangliomas in NSG mice provide a potentially valuable new tool that could prove especially valuable for testing treatments aimed at eradicating the small tumor deposits that are often numerous in patients with metastatic paraganglioma.

Cytocidal activities of topoisomerase 1 inhibitors and 5-azacytidine against pheochromocytoma/paraganglioma cells in primary human tumor cultures and mouse cell lines.

There is currently no effective treatment for metastatic pheochromocytomas and paragangliomas. A deficiency in current chemotherapy regimens is that the metastases usually grow very slowly. Drugs that target dividing tumor cells have therefore had limited success. To improve treatment, new strategies and valid experimental models are required for pre-clinical testing. However, development of models has itself been hampered by the absence of human pheochromocytoma/paraganglioma cell lines for cultures or xenografts. Topoisomerase 1 (TOP1) inhibitors are drugs that interfere with mechanisms that maintain DNA integrity during transcription in both quiescent and dividing cells. We used primary cultures of representative human tumors to establish the cytotoxicity of camptothecin, a prototypical TOP1 inhibitor, against non-dividing pheochromocytoma/paraganglioma cells, and then employed a mouse pheochromocytoma model (MPC) to show that efficacy of low concentrations of camptothecin and other TOP1 inhibitors is increased by intermittent coadministration of sub-toxic concentrations of 5-azacytidine, a DNA methylation inhibitor that modulates transcription. We then tested the same drugs against a clonal MPC derivative that expresses CMV reporter-driven luciferase and GFP, intended for in vivo drug testing. Unexpectedly, luciferase expression, bioluminescence and GFP expression were paradoxically increased by both camptothecin and SN38, the active metabolite of irinotecan, thereby masking cell death. Expression of chromogranin A, a marker for neuroendocrine secretory granules, was not increased, indicating that the drug effects on levels of luciferase and GFP are specific to the GFP-luciferase construct rather than generalized cellular responses. Our findings provide proof of principle for use of TOP1 inhibitors against pheochromocytoma/paraganglioma and suggest novel strategies for enhancing efficacy and reducing toxicity by optimizing the combination and timing of their use in conjunction with other drugs. The paradoxical effects of TOP1 inhibitors on luciferase and GFP dictate a need for caution in the use of CMV promoter-regulated constructs for cancer-related imaging studies.

Landscape of the mitochondrial Hsp90 metabolome in tumours.

Reprogramming of tumour cell metabolism contributes to disease progression and resistance to therapy, but how this process is regulated on the molecular level is unclear. Here we report that heat shock protein 90-directed protein folding in mitochondria controls central metabolic networks in tumour cells, including the electron transport chain, citric acid cycle, fatty acid oxidation, amino acid synthesis and cellular redox status. Specifically, mitochondrial heat shock protein 90, but not cytosolic heat shock protein 90, binds and stabilizes the electron transport chain Complex II subunit succinate dehydrogenase-B, maintaining cellular respiration under low-nutrient conditions, and contributing to hypoxia-inducible factor-1α-mediated tumorigenesis in patients carrying succinate dehydrogenase-B mutations. Thus, heat shock protein 90-directed proteostasis in mitochondria regulates tumour cell metabolism, and may provide a tractable target for cancer therapy.

Genotype and tumor locus determine expression profile of pseudohypoxic pheochromocytomas and paragangliomas.

Pheochromocytomas (PHEOs) and paragangliomas (PGLs) related to mutations in the mitochondrial succinate dehydrogenase (SDH) subunits A, B, C, and D, SDH complex assembly factor 2, and the von Hippel-Lindau (VHL) genes share a pseudohypoxic expression profile. However, genotype-specific differences in expression have been emerging. Development of effective new therapies for distinctive manifestations, e.g., a high rate of malignancy in SDHB- or predisposition to multifocal PGLs in SDHD patients, mandates improved stratification. To identify mutation/location-related characteristics among pseudohypoxic PHEOs/PGLs, we used comprehensive microarray profiling (SDHB: n = 18, SDHD-abdominal/thoracic (AT): n = 6, SDHD-head/neck (HN): n = 8, VHL: n = 13). To avoid location-specific bias, typical adrenal medulla genes were derived from matched normal medullas and cortices (n = 8) for data normalization. Unsupervised analysis identified two dominant clusters, separating SDHB and SDHD-AT PHEOs/PGLs (cluster A) from VHL PHEOs and SDHD-HN PGLs (cluster B). Supervised analysis yielded 6937 highly predictive genes (misclassification error rate of 0.175). Enrichment analysis revealed that energy metabolism and inflammation/fibrosis-related genes were most pronouncedly changed in clusters A and B, respectively. A minimum subset of 40 classifiers was validated by quantitative real-time polymerase chain reaction (quantitative real-time polymerase chain reaction vs. microarray: r = 0.87). Expression of several individual classifiers was identified as characteristic for VHL and SDHD-HN PHEOs and PGLs. In the present study, we show for the first time that SDHD-HN PGLs share more features with VHL PHEOs than with SDHD-AT PGLs. The presented data suggest novel subclassification of pseudohypoxic PHEOs/PGLs and implies cluster-specific pathogenic mechanisms and treatment strategies.

Combined inhibition of mTORC1 and mTORC2 signaling pathways is a promising therapeutic option in inhibiting pheochromocytoma tumor growth: in vitro and in vivo studies in female athymic nude mice.

Several lines of evidence, including the recent discovery of novel susceptibility genes, point out an important role for the mammalian target of rapamycin (mTOR) signaling pathway in the development of pheochromocytoma. Analyzing a set of pheochromocytomas from patients with different genetic backgrounds, we observed and confirmed a significant overexpression of key mTOR complex (mTORC) signaling mediators. Using selective ATP-competitive inhibitors targeting both mTORC1 and mTORC2, we significantly arrested the in vitro cell proliferation and blocked migration of pheochromocytoma cells as a result of the pharmacological suppression of the Akt/mTOR signaling pathway. Moreover, AZD8055, a selective ATP-competitive dual mTORC1/2 small molecular inhibitor, significantly reduced the tumor burden in a model of metastatic pheochromocytoma using female athymic nude mice. This study suggests that targeting both mTORC1 and mTORC2 is a potentially rewarding strategy and supports the application of selective inhibitors in combinatorial drug regimens for metastatic pheochromocytoma.

Characterization of two mouse models of metastatic pheochromocytoma using bioluminescence imaging.

Pheochromocytoma is the most common tumor of the adrenal medulla in adults. The lack of sensitive animal models of pheochromocytoma has hindered the study of this tumor and in vivo evaluation of antitumor agents. In this study we generated two sensitive luciferase models using bioluminescent pheochromocytoma cells: an experimental metastasis model to monitor tumor spreading and a subcutaneous model to monitor tumor growth and spontaneous metastasis. These models offer a platform for sensitive, non-invasive and real-time monitoring of pheochromocytoma primary growth and metastatic burden to follow the course of tumor progression and for testing relevant antitumor treatments in metastatic pheochromocytoma.

Usefulness of [18F]-DA and [18F]-DOPA for PET imaging in a mouse model of pheochromocytoma.

PURPOSE: To evaluate the usefulness of [(18)F]-6-fluorodopamine ([(18)F]-DA) and [(18)F]-L-6-fluoro-3,4-dihydroxyphenylalanine ([(18)F]-DOPA) positron emission tomography (PET) in the detection of subcutaneous (s.c.) and metastatic pheochromocytoma in mice; to assess the expression of the norepinephrine transporter (NET) and vesicular monoamine transporters 1 and 2 (VMAT1 and VMAT2), all important for [(18)F]-DA and [(18)F]-DOPA uptake. Furthermore, to compare tumor detection by micro-computed tomography (microCT) to magnetic resonance imaging (MRI) in individual mouse. METHODS: SUV(max) values were calculated from [(18)F]-DA and [(18)F]-DOPA PET, tumor-to-liver ratios (TLR) were obtained and expression of NET, VMAT1 and VMAT2 was evaluated. RESULTS: [(18)F]-DA detected less metastatic lesions compared to [(18)F]-DOPA. TLR values for liver metastases were 2.26-2.71 for [(18)F]-DOPA and 1.83-2.83 for [(18)F]-DA. A limited uptake of [(18)F]-DA was found in s.c. tumors (TLR = 0.22-0.27) compared to [(18)F]-DOPA (TLR = 1.56-2.24). Overall, NET and VMAT2 were expressed in all organ and s.c. tumors. However, s.c. tumors lacked expression of VMAT1. We confirmed [(18)F]-DA's high affinity for the NET for its uptake and VMAT1 and VMAT2 for its storage and retention in pheochromocytoma cell vesicles. In contrast, [(18)F]-DOPA was found to utilize only VMAT2. CONCLUSION: MRI was superior in the detection of all organ tumors compared to microCT and PET. [(18)F]-DOPA had overall better sensitivity than [(18)F]-DA for the detection of metastases. Subcutaneous tumors were localized only with [(18)F]-DOPA, a finding that may reflect differences in expression of VMAT1 and VMAT2, perhaps similar to some patients with pheochromocytoma where [(18)F]-DOPA provides better visualization of lesions than [(18)F]-DA.

Phosphorylation state of Olig2 regulates proliferation of neural progenitors.

The bHLH transcription factors that regulate early development of the central nervous system can generally be classified as either antineural or proneural. Initial expression of antineural factors prevents cell cycle exit and thereby expands the pool of neural progenitors. Subsequent (and typically transient) expression of proneural factors promotes cell cycle exit, subtype specification, and differentiation. Against this backdrop, the bHLH transcription factor Olig2 in the oligodendrocyte lineage is unorthodox, showing antineural functions in multipotent CNS progenitor cells but also sustained expression and proneural functions in the formation of oligodendrocytes. We show here that the proliferative function of Olig2 is controlled by developmentally regulated phosphorylation of a conserved triple serine motif within the amino-terminal domain. In the phosphorylated state, Olig2 maintains antineural (i.e., promitotic) functions that are reflected in human glioma cells and in a genetically defined murine model of primary glioma.

Tyrosine hydroxylase, chromogranin A, and steroidogenic acute regulator as markers for successful separation of human adrenal medulla.

Progress in high throughput "-omic" techniques now allows the simultaneous measurement of expression levels of thousands of genes and promises the improved understanding of the molecular biology of diseases such as cancer. Detection of the dysfunction of molecular pathways in diseases requires healthy control tissue. This is difficult to obtain from pheochromocytomas (PHEOs), rare chromaffin tumors derived from adrenal medulla. The two options for obtaining adrenal tissue are: (1) whole organ removal post-mortem or during radical nephrectomy; (2) removal during PHEO surgery. Access to high quality normal adrenal tissue is limited. Removal of whole adrenals during nephrectomy is rare, because of improved surgical techniques. For adrenals removed post-mortem, the lag time to proper organ perfusion causes uncontrolled tissue degradation. Adjacent normal adrenal tissue can almost never be obtained from resected PHEOs, because they often replace the entire medulla or are well-encapsulated. If a margin of normal adrenal is attached to a resected PHEO, it seldom contains any medulla. The clean separation of medulla and cortex is further complicated, because their border is convoluted, and because adult adrenal consists of approximately 90% cortex. Thus, the quality of separation has to be evaluated with specific medullary and cortical markers. We describe the successful dissection of highly pure, medullary tissue from adrenals snap-frozen upon resection during radical nephrectomy or after brain death. Separation quality has been verified by quantitative reverse transcription with polymerase chain reaction for the medullary enzymes, tyrosine hydroxylase, and chromogranin A, and for the cortical enzyme, steroidogenic acute regulator.

Noninvasive monitoring of a murine model of metastatic pheochromocytoma: a comparison of contrast-enhanced microCT and nonenhanced MRI.

PURPOSE: To compare contrast-enhanced micro-computed tomography (microCT) and nonenhanced respiratory-triggered magnetic resonance imaging (MRI) in an animal model of metastatic pheochromocytoma. Animal models are becoming important in the study of cancer treatment and imaging is useful in minimizing the number of animals needed and reducing costs associated with autopsies. However, the choice of imaging modality is still evolving. MATERIALS AND METHODS: Adult female nude mice were injected by tail vein with a mouse pheochromocytoma (MPC) cell line (MPC 4/30PRR) to create a metastatic model. After optimizing imaging techniques, eight mice were imaged with both respiratory triggered MRI and microCT and the findings were verified histologically. RESULTS: MicroCT and MRI were approximately equal in their ability to detect hepatic metastases at a size threshold of 350 microm. In the lungs, MRI was more sensitive than microCT, detecting lesions 0.6 mm in diameter versus 1 mm for microCT. Additionally, MRI was more sensitive for lesions in the kidneys, bone, ovaries, and adrenal glands. MRI demonstrated a higher contrast-to-noise ratio (CNR) than microCT. CONCLUSION: In addition to the advantage of not exposing the animal to ionizing radiation, MRI provided a more complete assessment of the extent of metastases in this model compared to microCT.

Oncocytic adrenal cortical tumor with cytoplasmic inclusions and hyaline globules.

Adrenal cortical tumors, particularly oncocytic tumors, have been reported to contain a variety of intracytoplasmic and intramitochondrial inclusions. Oncocytic cortical tumors can also morphologically mimic pheochromocytomas. We report an unusual, partially oncocytic cortical neoplasm with nesting architecture, intranuclear inclusions, and hyaline globules reminiscent of pheochromocytoma, together with numerous, small, brightly eosinophilic, periodic acid-Schiff-positive cytoplasmic inclusions and typical cytoplasmic lipid droplets. Ultrastructural study revealed oncocytes containing numerous mitochondria with intramitochondrial crystals and lipid droplets. Immunohistochemistry and immunoblots were utilized to further characterize the tumor. Immunohistochemistry demonstrated immunoreactivity of both the eosinophilic inclusions and the hyaline globules for adipose differentiation-related protein (ADRP), which is one of a group of proteins associated with storage of neutral lipids in many cell types. Immunoblots confirmed the presence of ADRP and demonstrated an imbalance between ADRP and perilipin, another neutral lipid-associated protein, in tumor tissue compared to normal adrenal cortex. The findings suggest that mitochondrial dysfunction in oncocytic cortical tumors may lead to abnormal processing of proteins related to the lipid-storing functions of the adrenal cortex, resulting in unusual cytoplasmic inclusions and extracellular globules resembling the globules in pheochromocytomas. The finding of ADRP as a constituent of inclusions in adrenal cortical tumors has not been previously reported.