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2.
Nat Metab ; 4(6): 739-758, 2022 06.
Article in English | MEDLINE | ID: mdl-35760869

ABSTRACT

Mitochondria are the main consumers of oxygen within the cell. How mitochondria sense oxygen levels remains unknown. Here we show an oxygen-sensitive regulation of TFAM, an activator of mitochondrial transcription and replication, whose alteration is linked to tumours arising in the von Hippel-Lindau syndrome. TFAM is hydroxylated by EGLN3 and subsequently bound by the von Hippel-Lindau tumour-suppressor protein, which stabilizes TFAM by preventing mitochondrial proteolysis. Cells lacking wild-type VHL or in which EGLN3 is inactivated have reduced mitochondrial mass. Tumorigenic VHL variants leading to different clinical manifestations fail to bind hydroxylated TFAM. In contrast, cells harbouring the Chuvash polycythaemia VHLR200W mutation, involved in hypoxia-sensing disorders without tumour development, are capable of binding hydroxylated TFAM. Accordingly, VHL-related tumours, such as pheochromocytoma and renal cell carcinoma cells, display low mitochondrial content, suggesting that impaired mitochondrial biogenesis is linked to VHL tumorigenesis. Finally, inhibiting proteolysis by targeting LONP1 increases mitochondrial content in VHL-deficient cells and sensitizes therapy-resistant tumours to sorafenib treatment. Our results offer pharmacological avenues to sensitize therapy-resistant VHL tumours by focusing on the mitochondria.


Subject(s)
Carcinoma, Renal Cell , Kidney Neoplasms , von Hippel-Lindau Disease , ATP-Dependent Proteases , Carcinoma, Renal Cell/genetics , Humans , Kidney Neoplasms/genetics , Mitochondrial Proteins , Organelle Biogenesis , Oxygen , von Hippel-Lindau Disease/genetics
3.
Nucleic Acids Res ; 49(5): 2509-2521, 2021 03 18.
Article in English | MEDLINE | ID: mdl-33555349

ABSTRACT

The paucity of recurrent mutations has hampered efforts to understand and treat neuroblastoma. Alternative splicing and splicing-dependent RNA-fusions represent mechanisms able to increase the gene product repertoire but their role in neuroblastoma remains largely unexplored. Here we investigate the presence and possible roles of aberrant splicing and splicing-dependent RNA-fusion transcripts in neuroblastoma. In addition, we attend to establish whether the spliceosome can be targeted to treat neuroblastoma. Through analysis of RNA-sequenced neuroblastoma we show that elevated expression of splicing factors is a strong predictor of poor clinical outcome. Furthermore, we identified >900 primarily intrachromosomal fusions containing canonical splicing sites. Fusions included transcripts from well-known oncogenes, were enriched for proximal genes and in chromosomal regions commonly gained or lost in neuroblastoma. As a proof-of-principle that these fusions can generate altered gene products, we characterized a ZNF451-BAG2 fusion, producing a truncated BAG2-protein which inhibited retinoic acid induced differentiation. Spliceosome inhibition impeded neuroblastoma fusion expression, induced apoptosis and inhibited xenograft tumor growth. Our findings elucidate a splicing-dependent mechanism generating altered gene products in neuroblastoma and show that the spliceosome is a potential target for clinical intervention.


Subject(s)
Molecular Chaperones/genetics , Mutant Chimeric Proteins/genetics , Neuroblastoma/genetics , RNA Splicing , Spliceosomes/drug effects , Aminoacyltransferases/metabolism , Animals , Apoptosis , Cell Differentiation , Cell Line, Tumor , Female , Gene Fusion , HSC70 Heat-Shock Proteins/metabolism , Humans , Mice, Nude , Molecular Chaperones/metabolism , Mutant Chimeric Proteins/metabolism , Neuroblastoma/metabolism , Neuroblastoma/pathology , RNA Splicing Factors/genetics , RNA Splicing Factors/metabolism , Sequence Deletion , Transcription Factors/metabolism , tau Proteins/metabolism
4.
Int J Mol Sci ; 21(12)2020 Jun 16.
Article in English | MEDLINE | ID: mdl-32560271

ABSTRACT

Solid tumors, including breast cancer, are characterized by the hypoxic microenvironment, extracellular acidosis, and chemoresistance. Hypoxia marker, carbonic anhydrase IX (CAIX), is a pH regulator providing a selective survival advantage to cancer cells through intracellular neutralization while facilitating tumor invasion by extracellular acidification. The expression of CAIX in breast cancer patients is associated with poor prognosis and metastases. Importantly, CAIX-positive hypoxic tumor regions are enriched in cancer stem cells (CSCs). Here we investigated the biological effects of CA9-silencing in breast cancer cell lines. We found that CAIX-downregulation in hypoxia led to increased levels of let-7 (lethal-7) family members. Simultaneously with the increase of let-7 miRNAs in CAIX-suppressed cells, LIN28 protein levels decreased, along with downstream metabolic pathways: pyruvate dehydrogenase kinase 1 (PDK1) and phosphorylation of its substrate, pyruvate dehydrogenase (PDH) at Ser-232, causing attenuation of glycolysis. In addition to perturbed glycolysis, CAIX-knockouts, in correlation with decreased LIN28 (as CSC reprogramming factor), also exhibit reduction of the further CSC-associated markers NANOG (Homeobox protein NANOG) and ALDH1 (Aldehyde dehydrogenase isoform 1). Oppositely, overexpression of CAIX leads to the enhancement of LIN28, ALDH1, and NANOG. In conclusion, CAIX-driven regulation of the LIN28/let-7 axis augments glycolytic metabolism and enhances stem cell markers expression during CAIX-mediated adaptation to hypoxia and acidosis in carcinogenesis.


Subject(s)
Antigens, Neoplasm/genetics , Breast Neoplasms/metabolism , Carbonic Anhydrase IX/genetics , MicroRNAs/genetics , Neoplastic Stem Cells/metabolism , RNA-Binding Proteins/genetics , Breast Neoplasms/genetics , Cell Hypoxia , Cell Line, Tumor , Cellular Reprogramming , Female , Gene Expression Profiling , Glycolysis , Humans , Hydrogen-Ion Concentration , MCF-7 Cells
5.
Aging (Albany NY) ; 12(7): 5781-5791, 2020 04 01.
Article in English | MEDLINE | ID: mdl-32235007

ABSTRACT

Hypoxia-inducible factors (HIFs) regulate oxygen sensing and expression of genes involved in angiogenesis and erythropoiesis. Polycythemia has been observed in patients with hepatocellular carcinoma (HCC), but the underlying molecular basis remains unknown. Liver tissues from 302 HCC patients, including 104 with polycythemia, were sequenced for HIF2A mutations. A germline HIF2A mutation was detected in one HCC patient with concurrent polycythemia. Three additional family members carried this mutation, but none exhibited polycythemia or were diagnosed with HCC. The gain-of-function mutation resulted in a HIF-2α protein that was transcribed normally but resistant to degradation. HIF-2α target genes EDN1, EPO, GNA14, and VEGF were significantly upregulated in the tumor bed but not in the surrounding liver tissue. Polycythemia resolved upon total resection of the tumor tissue. This newly described HIF2A mutation may promote HCC oncogenesis.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Carcinoma, Hepatocellular/genetics , Gain of Function Mutation , Germ-Line Mutation , Liver Neoplasms/genetics , Polycythemia/genetics , Adult , Carcinoma, Hepatocellular/pathology , DNA Mutational Analysis , Humans , Liver Neoplasms/pathology , Male , Polycythemia/pathology
6.
Proc Natl Acad Sci U S A ; 116(34): 16997-17006, 2019 08 20.
Article in English | MEDLINE | ID: mdl-31375625

ABSTRACT

Despite the discovery of the oxygen-sensitive regulation of HIFα by the von Hippel-Lindau (VHL) protein, the mechanisms underlying the complex genotype/phenotype correlations in VHL disease remain unknown. Some germline VHL mutations cause familial pheochromocytoma and encode proteins that preserve their ability to down-regulate HIFα. While type 1, 2A, and 2B VHL mutants are defective in regulating HIFα, type 2C mutants encode proteins that preserve their ability to down-regulate HIFα. Here, we identified an oxygen-sensitive function of VHL that is abolished by VHL type 2C mutations. We found that BIM-EL, a proapoptotic BH3-only protein, is hydroxylated by EglN3 and subsequently bound by VHL. VHL mutants fail to bind hydroxylated BIM-EL, regardless of whether they have the ability to bind hydroxylated HIFα or not. VHL binding inhibits BIM-EL phosphorylation by extracellular signal-related kinase (ERK) on serine 69. This causes BIM-EL to escape from proteasomal degradation, allowing it to enhance EglN3-induced apoptosis. BIM-EL was rapidly degraded in cells lacking wild-type VHL or in which EglN3 was inactivated genetically or by lack of oxygen, leading to enhanced cell survival and chemotherapy resistance. Combination therapy using ERK inhibitors, however, resensitizes VHL- and EglN3-deficient cells that are otherwise cisplatin-resistant.


Subject(s)
Adrenal Gland Neoplasms , Bcl-2-Like Protein 11/metabolism , Drug Resistance, Neoplasm/genetics , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Mutation , Pheochromocytoma , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Adrenal Gland Neoplasms/drug therapy , Adrenal Gland Neoplasms/genetics , Adrenal Gland Neoplasms/metabolism , Adrenal Gland Neoplasms/pathology , Animals , Apoptosis/drug effects , Apoptosis/genetics , Bcl-2-Like Protein 11/genetics , Cell Hypoxia/drug effects , Cell Hypoxia/genetics , Cisplatin/pharmacology , Drug Resistance, Neoplasm/drug effects , Humans , Hydroxylation/drug effects , Hydroxylation/genetics , Hypoxia-Inducible Factor-Proline Dioxygenases/genetics , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , Mice , Mice, Knockout , PC12 Cells , Pheochromocytoma/drug therapy , Pheochromocytoma/metabolism , Pheochromocytoma/pathology , Proteolysis/drug effects , Rats , Von Hippel-Lindau Tumor Suppressor Protein/genetics
7.
Clin Cancer Res ; 24(14): 3423-3432, 2018 07 15.
Article in English | MEDLINE | ID: mdl-29636359

ABSTRACT

Purpose: Cluster I pheochromocytomas and paragangliomas (PCPGs) tend to develop malignant transformation, tumor recurrence, and multiplicity. Transcriptomic profiling suggests that cluster I PCPGs and other related tumors exhibit distinctive changes in the tricarboxylic acid (TCA) cycle, the hypoxia signaling pathway, mitochondrial electron transport chain, and methylation status, suggesting that therapeutic regimen might be optimized by targeting these signature molecular pathways.Experimental Design: In the present study, we investigated the molecular signatures in clinical specimens from cluster I PCPGs in comparison with cluster II PCPGs that are related to kinase signaling and often present as benign tumors.Results: We found that cluster I PCPGs develop a dependency to mitochondrial complex I, evidenced by the upregulation of complex I components and enhanced NADH dehydrogenation. Alteration in mitochondrial function resulted in strengthened NAD+ metabolism, here considered as a key mechanism of chemoresistance, particularly, of succinate dehydrogenase subunit B (SDHB)-mutated cluster I PCPGs via the PARP1/BER DNA repair pathway. Combining a PARP inhibitor with temozolomide, a conventional chemotherapeutic agent, not only improved cytotoxicity but also reduced metastatic lesions, with prolonged overall survival of mice with SDHB knockdown PCPG allograft.Conclusions: In summary, our findings provide novel insights into an effective strategy for targeting cluster I PCPGs, especially those with SDHB mutations. Clin Cancer Res; 24(14); 3423-32. ©2018 AACR.


Subject(s)
DNA Repair , NAD/metabolism , Paraganglioma/genetics , Paraganglioma/metabolism , Pheochromocytoma/genetics , Pheochromocytoma/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Succinate Dehydrogenase/genetics , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Apoptosis/genetics , Cell Cycle/genetics , Cell Line, Tumor , Disease Models, Animal , Drug Resistance, Neoplasm/genetics , Humans , Mice , Mitochondria/metabolism , Models, Biological , Molecular Targeted Therapy , Mutation , Paraganglioma/drug therapy , Paraganglioma/pathology , Pheochromocytoma/drug therapy , Pheochromocytoma/pathology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Signal Transduction/drug effects , Xenograft Model Antitumor Assays
8.
Oncotarget ; 8(34): 56110-56125, 2017 Aug 22.
Article in English | MEDLINE | ID: mdl-28915577

ABSTRACT

Histone deacetylase inhibitors (HDACis) are a potent class of tumor-suppressive agents traditionally believed to exert their effects through loosening tightly-wound chromatin resulting in de-inhibition of various tumor suppressive genes. Recent literature however has shown altered intratumoral hypoxia signaling with HDACi administration not attributable to changes in chromatin structure. We sought to determine the precise mechanism of HDACi-mediated hypoxia signaling attenuation using vorinostat (SAHA), an FDA-approved class I/IIb/IV HDACi. Through an in-vitro and in-vivo approach utilizing cell lines for hepatocellular carcinoma (HCC), osteosarcoma (OS), and glioblastoma (GBM), we demonstrate that SAHA potently inhibits HIF-a nuclear translocation via direct acetylation of its associated chaperone, heat shock protein 90 (Hsp90). In the presence of SAHA we found elevated levels of acetyl-Hsp90, decreased interaction between acetyl-Hsp90 and HIF-a, decreased nuclear/cytoplasmic HIF-α expression, absent HIF-α association with its nuclear karyopharyin Importin, and markedly decreased HIF-a transcriptional activity. These changes were associated with downregulation of downstream hypoxia molecules such as endothelin 1, erythropoietin, glucose transporter 1, and vascular endothelial growth factor. Findings were replicated in an in-vivo Hep3B HRE-Luc expressing xenograft, and were associated with significant decreases in xenograft tumor size. Altogether, this study highlights a novel mechanism of action of an important class of chemotherapeutic.

9.
Endocrinology ; 158(10): 3097-3108, 2017 10 01.
Article in English | MEDLINE | ID: mdl-28938421

ABSTRACT

Proteasome inhibitors have been frequently used in treating hematologic and solid tumors. They are administered individually or in combination with other regimens, to prevent severe side effects and resistance development. Because they have been shown to be efficient and are pharmaceutically available, we tested the first Food and Drug Administration-approved proteasome inhibitor bortezomib alone and in combination with another proteasome inhibitor, salinosporamid A, in pheochromocytoma cells. Pheochromocytomas/Paragangliomas (PHEOs/PGLs) are neuroendocrine tumors for which no definite cure is yet available. Therefore, drugs with a wide spectrum of mechanisms of action are being tested to identify suitable candidates for PHEO/PGL treatment. In the current study, we show that bortezomib induces PHEO cell death via the apoptotic pathway in vitro and in vivo. The combination of bortezomib with salinosporamid A exhibits additive effect on these cells and inhibits proliferation, cell migration and invasion, and angiogenesis more potently than bortezomib alone. Altogether, we suggest these proteasome inhibitors, especially bortezomib, could be potentially tested in PHEO/PGL patients who might benefit from treatment with either the inhibitors alone or in combination with other treatment options.


Subject(s)
Adrenal Gland Neoplasms/pathology , Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Bortezomib/administration & dosage , Lactones/administration & dosage , Pheochromocytoma/pathology , Pyrroles/administration & dosage , Adrenal Gland Neoplasms/drug therapy , Animals , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Female , Mice , Mice, Nude , Neoplasm Invasiveness/prevention & control , Neovascularization, Pathologic/prevention & control , Pheochromocytoma/drug therapy , Proteasome Inhibitors/administration & dosage
10.
Oncotarget ; 8(14): 22313-22324, 2017 Apr 04.
Article in English | MEDLINE | ID: mdl-28423608

ABSTRACT

Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are rare, neuroendocrine tumors derived from adrenal or extra-adrenal chromaffin cells, respectively. Metastases are discovered in 3-36% of patients at the time of diagnosis. Currently, only suboptimal treatment options exist. Therefore, new therapeutic compounds targeting metastatic PHEOs/PGLs are urgently needed. Here, we investigated if anthracyclines were able to suppress the progression of metastatic PHEO. We explored their effects on experimental mouse PHEO tumor cells using in vitro and in vivo models, and demonstrated that anthracyclines, particularly idarubicin (IDA), suppressed hypoxia signaling by preventing the binding of hypoxia-inducible factor 1 and 2 (HIF-1 and HIF-2) to the hypoxia response element (HRE) sites on DNA. This resulted in reduced transcriptional activation of HIF target genes, including erythropoietin (EPO), phosphoglycerate kinase 1 (PGK1), endothelin 1 (EDN1), glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and vascular endothelial growth factor (VEGFA), which consequently inhibited the growth of metastatic PHEO. Additionally, IDA downregulated hypoxia signaling by interfering with the transcriptional activation of HIF1A and HIF2A. Furthermore, our animal model demonstrated the dose-dependent suppressive effect of IDA on metastatic PHEO growth in vivo. Our results indicate that anthracyclines are prospective candidates for inclusion in metastatic PHEO/PGL therapy, especially in patients with gene mutations involved in the hypoxia signaling pathway.


Subject(s)
Adrenal Gland Neoplasms/drug therapy , Antineoplastic Agents/therapeutic use , Hypoxia/drug therapy , Idarubicin/therapeutic use , Pheochromocytoma/drug therapy , Adrenal Gland Neoplasms/pathology , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Growth Processes/drug effects , Cell Line, Tumor , Endothelin-1/genetics , Endothelin-1/metabolism , Erythropoietin/genetics , Erythropoietin/metabolism , Humans , Hypoxia/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Mice, Nude , Neoplasm Metastasis , Pheochromocytoma/pathology , Phosphoglycerate Kinase/genetics , Phosphoglycerate Kinase/metabolism , Protein Binding , Signal Transduction/drug effects , Xenograft Model Antitumor Assays
11.
Oncotarget ; 7(26): 40531-40545, 2016 Jun 28.
Article in English | MEDLINE | ID: mdl-27244895

ABSTRACT

Hypoxia is a common feature of solid tumors that activates a plethora of pathways, resulting in proliferation and resistance of cancer cells to radio- and chemotherapy. Pheochromocytomas/paragangliomas (PHEOs/PGLs) with mutations in the gene coding for the subunit B of succinate dehydrogenase (SDHB) are the most aggressive forms of the disease, which is partially due to their pseudohypoxic character, metabolic abnormalities, and elevated reactive oxygen species (ROS) levels. We investigated the effect of piperlongumine (PL), a natural product with cytotoxic properties restricted to cancer cells by significantly increasing intracellular ROS levels, on PHEO cells. Here we report for the first time that PL mediates PHEO cell death by activating both apoptosis and necroptosis in vitro and in vivo. This effect is magnified in hypoxic conditions, making PL a promising potential candidate for use as a therapeutic option for patients with PHEO/PGL, including those with SDHB mutations.


Subject(s)
Adrenal Gland Neoplasms/drug therapy , Dioxolanes/pharmacology , Hypoxia , Paraganglioma/drug therapy , Pheochromocytoma/drug therapy , Adrenal Gland Neoplasms/genetics , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Cell Death , Cell Line, Tumor , Cell Movement , Cell Survival , Female , Humans , Mice , Mice, Nude , Neoplasm Invasiveness , Oxidation-Reduction , Paraganglioma/genetics , Pheochromocytoma/genetics , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolism , Succinate Dehydrogenase/genetics
13.
Endocr Pract ; 22(2): 190-5, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26492543

ABSTRACT

OBJECTIVE: To describe a patient with a germline succinate dehydrogenase (SDHC) gene mutation presenting with primary hyperparathyroidism and a large catecholamine-producing temporal bone paraganglioma (PGL). METHODS: Evaluation of a SDHC mutation-positive PGL tumor biology using staining for tyrosine hydroxylase (TH), hypoxia-inducible factors 1α (HIF-1α) and 2α (HIF-2α). RESULTS: A 66-year-old man was noted to have a lytic skull base mass during work-up for his primary hyperparathyroidism. Biochemical evaluation with 24-hour urine catecholamines and metanephrines revealed marked elevation of norepinephrine and normetanephrine. Genetic testing revealed a germline SDHC mutation. A partial excision of skull base tumor was performed, which upon further examination revealed PGL. Immunohistochemistry of skull base PGL demonstrated heavy expression of TH and HIF-2α but reduced expression of HIF-1α. The remaining skull base PGL was treated with adjuvant radiation therapy. The patient's normetanephrine levels significantly decreased after surgery and radiation. CONCLUSION: Here, we report an unusual case of a patient presenting with a germline SDHC mutation-related functional PGL along with concomitant primary hyperparathyroidism. The present case illustrates that overexpression of HIF-2α but not of HIF-1α is linked to the pathogenesis of SDHC mutation-related PGL, and it may be responsible for the aggressive clinical behavior of a usually indolent course of SDHC-related PGLs.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Membrane Proteins/genetics , Paraganglioma/pathology , Skull Neoplasms/pathology , Temporal Bone/pathology , Aged , Germ-Line Mutation , Humans , Male , Neoplasm Invasiveness , Paraganglioma/diagnostic imaging , Paraganglioma/genetics , Paraganglioma/metabolism , Radionuclide Imaging , Signal Transduction , Skull Neoplasms/diagnostic imaging , Skull Neoplasms/genetics , Skull Neoplasms/metabolism , Up-Regulation
14.
Endocrinology ; 156(11): 4094-104, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26267380

ABSTRACT

Metastatic pheochromocytoma continues to be an incurable disease, and treatment with conventional cytotoxic chemotherapy offers limited efficacy. In the present study, we evaluated a novel topoisomerase I inhibitor, LMP-400, as a potential treatment for this devastating disease. We found a high expression of topoisomerase I in human metastatic pheochromocytoma, providing a basis for the evaluation of a topoisomerase 1 inhibitor as a therapeutic strategy. LMP-400 inhibited the cell growth of established mouse pheochromocytoma cell lines and primary human tumor tissue cultures. In a study performed in athymic female mice, LMP-400 demonstrated a significant inhibitory effect on tumor growth with two drug administration regimens. Furthermore, low doses of LMP-400 decreased the protein levels of hypoxia-inducible factor 1 (HIF-1α), one of a family of factors studied as potential metastatic drivers in these tumors. The HIF-1α decrease resulted in changes in the mRNA levels of HIF-1 transcriptional targets. In vitro, LMP-400 showed an increase in the growth-inhibitory effects in combination with other chemotherapeutic drugs that are currently used for the treatment of pheochromocytoma. We conclude that LMP-400 has promising antitumor activity in preclinical models of metastatic pheochromocytoma and its use should be considered in future clinical trials.


Subject(s)
Adrenal Gland Neoplasms/drug therapy , Benzodioxoles/pharmacology , Isoquinolines/pharmacology , Pheochromocytoma/drug therapy , Topoisomerase I Inhibitors/pharmacology , Adrenal Gland Neoplasms/enzymology , Adrenal Gland Neoplasms/pathology , Animals , Antineoplastic Agents/pharmacology , Benzodioxoles/administration & dosage , Blotting, Western , Cell Hypoxia , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Topoisomerases, Type I/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Synergism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Isoquinolines/administration & dosage , Liver Neoplasms/drug therapy , Liver Neoplasms/enzymology , Liver Neoplasms/secondary , Lung Neoplasms/drug therapy , Lung Neoplasms/enzymology , Lung Neoplasms/secondary , Mice, Nude , PC12 Cells , Pheochromocytoma/enzymology , Pheochromocytoma/pathology , Rats , Reverse Transcriptase Polymerase Chain Reaction , Topoisomerase I Inhibitors/administration & dosage , Tumor Cells, Cultured
15.
J Clin Endocrinol Metab ; 100(5): E710-9, 2015 May.
Article in English | MEDLINE | ID: mdl-25695889

ABSTRACT

CONTEXT: Germline mutations in genes coding succinate dehydrogenase (SDH) subunits A, B, C, and D have been identified in familial paragangliomas (PGLs)/pheochromocytomas (PHEOs) and other tumors. We described a GH-secreting pituitary adenoma (PA) caused by SDHD mutation in a patient with familial PGLs. Additional patients with PAs and SDHx defects have since been reported. DESIGN: We studied 168 patients with unselected sporadic PA and with the association of PAs, PGLs, and/or pheochromocytomas, a condition we named the 3P association (3PAs) for SDHx germline mutations. We also studied the pituitary gland and hormonal profile of Sdhb(+/-) mice and their wild-type littermates at different ages. RESULTS: No SDHx mutations were detected among sporadic PA, whereas three of four familial cases were positive for a mutation (75%). Most of the SDHx-deficient PAs were either prolactinomas or somatotropinomas. Pituitaries of Sdhb(+/-) mice older than 12 months had an increased number mainly of prolactin-secreting cells and several ultrastructural abnormalities such as intranuclear inclusions, altered chromatin nuclear pattern, and abnormal mitochondria. Igf-1 levels of mutant mice tended to be higher across age groups, whereas Prl and Gh levels varied according to age and sex. CONCLUSION: The present study confirms the existence of a new association that we termed 3PAs. It is due mostly to germline SDHx defects, although sporadic cases of 3PAs without SDHx defects also exist. Using Sdhb(+/-) mice, we provide evidence that pituitary hyperplasia in SDHx-deficient cells may be the initial abnormality in the cascade of events leading to PA formation.


Subject(s)
Adenoma/genetics , Adrenal Gland Neoplasms/genetics , Paraganglioma/genetics , Pheochromocytoma/genetics , Pituitary Neoplasms/genetics , Succinate Dehydrogenase/genetics , Adolescent , Adult , Aged , Animals , Child , Child, Preschool , DNA Mutational Analysis , Female , Germ-Line Mutation , Humans , Male , Mice , Mice, Knockout , Middle Aged , Young Adult
16.
J Mol Med (Berl) ; 93(1): 93-104, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25263965

ABSTRACT

UNLABELLED: We have investigated genetic/pathogenetic factors associated with a new clinical entity in patients presenting with pheochromocytoma/paraganglioma (PHEO/PGL) and polycythemia. Two patients without hypoxia-inducible factor 2α (HIF2A) mutations, who presented with similar clinical manifestations, were analyzed for other gene mutations, including prolyl hydroxylase (PHD) mutations. We have found for the first time a germ-line mutation in PHD1 in one patient and a novel germ-line PHD2 mutation in a second patient. Both mutants exhibited reduced protein stability with substantial quantitative protein loss and thus compromised catalytic activities. Due to the unique association of patients' polycythemia with borderline or mildly elevated erythropoietin (EPO) levels, we also performed an in vitro sensitivity assay of erythroid progenitors to EPO and for EPO receptor (EPOR) expression. The results show inappropriate hypersensitivity of erythroid progenitors to EPO in these patients, indicating increased EPOR expression/activity. In addition, the present study indicates that HIF dysregulation due to PHD mutations plays an important role in the pathogenesis of these tumors and associated polycythemia. The PHD1 mutation appears to be a new member contributing to the genetic landscape of this novel clinical entity. Our results support the existence of a specific PHD1- and PHD2-associated PHEO/PGL-polycythemia disorder. KEY MESSAGE: • A novel germ-l i n e PHD1 mutation causing heochromocytoma/paraganglioma and polycythemia. • Increased EPOR activity and inappropriate hypersensitivity of erythroid progenitors to EPO.


Subject(s)
Hypoxia-Inducible Factor-Proline Dioxygenases/genetics , Pheochromocytoma/genetics , Polycythemia/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Female , Germ-Line Mutation , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Middle Aged , Pheochromocytoma/metabolism , Polycythemia/metabolism , Receptors, Erythropoietin/metabolism
17.
PLoS One ; 9(4): e90458, 2014.
Article in English | MEDLINE | ID: mdl-24699253

ABSTRACT

Drug repurposing or repositioning is an important part of drug discovery that has been growing in the last few years for the development of therapeutic options in oncology. We applied this paradigm in a screening of a library of about 3,800 compounds (including FDA-approved drugs and pharmacologically active compounds) employing a model of metastatic pheochromocytoma, the most common tumor of the adrenal medulla in children and adults. The collection of approved drugs was screened in quantitative mode, testing the compounds in compound-titration series (dose-response curves). Analysis of the dose-response screening data facilitated the selection of 50 molecules with potential bioactivity in pheochromocytoma cells. These drugs were classified based on molecular/cellular targets and signaling pathways affected, and selected drugs were further validated in a proliferation assay and by flow cytometric cell death analysis. Using meta-analysis information from molecular targets of the top drugs identified by our screening with gene expression data from human and murine microarrays, we identified potential drugs to be used as single drugs or in combination. An example of a combination with a synergistic effect is presented. Our study exemplifies a promising model to identify potential drugs from a group of clinically approved compounds that can more rapidly be implemented into clinical trials in patients with metastatic pheochromocytoma or paraganglioma.


Subject(s)
Adrenal Gland Neoplasms/drug therapy , Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Drug Discovery , Drug Repositioning , High-Throughput Screening Assays , Paraganglioma/drug therapy , Pheochromocytoma/drug therapy , Adrenal Gland Neoplasms/genetics , Adrenal Gland Neoplasms/pathology , Adult , Animals , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Flow Cytometry , Gene Expression Profiling , Humans , Mice , Oligonucleotide Array Sequence Analysis , Paraganglioma/genetics , Paraganglioma/pathology , Pheochromocytoma/genetics , Pheochromocytoma/secondary , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction
18.
Endocrinology ; 155(7): 2377-90, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24762141

ABSTRACT

Currently, there are no reliably effective therapeutic options for metastatic pheochromocytoma (PCC) and paraganglioma. Moreover, there are no therapies that may prevent the onset or progression of tumors in patients with succinate dehydrogenase type B mutations, which are associated with very aggressive tumors. Therefore, we tested the approved and well-tolerated drugs lovastatin and 13-cis-retinoic acid (13cRA) in vitro in an aggressive PCC mouse cell line, mouse tumor tissue-derived (MTT) cells, and in vivo in a PCC allograft nude mouse model, in therapeutically relevant doses. Treatment was started 24 hours before sc tumor cell injection and continued for 30 more days. Tumor sizes were measured from outside by caliper and sizes of viable tumor mass by bioluminescence imaging. Lovastatin showed antiproliferative effects in vitro and led to significantly smaller tumor sizes in vivo compared with vehicle treatment. 13cRA promoted tumor cell growth in vitro and led to significantly larger viable tumor mass and significantly faster increase of viable tumor mass in vivo over time compared with vehicle, lovastatin, and combination treatment. However, when combined with lovastatin, 13cRA enhanced the antiproliferative effect of lovastatin in vivo. The combination-treated mice showed slowest tumor growth of all groups with significantly slower tumor growth compared with the vehicle-treated mice and significantly smaller tumor sizes. Moreover, the combination-treated group displayed the smallest size of viable tumor mass and the slowest increase in viable tumor mass over time of all groups, with a significant difference compared with the vehicle- and 13cRA-treated group. The combination-treated tumors showed highest extent of necrosis, lowest median microvessel density and highest expression of α-smooth muscle actin. The combination of high microvessel density and low α-smooth muscle actin is a predictor of poor prognosis in other tumor entities. Therefore, this drug combination may be a well-tolerated novel therapeutic or preventive option for malignant PCC.


Subject(s)
Adrenal Gland Neoplasms/drug therapy , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Isotretinoin/pharmacology , Lovastatin/pharmacology , Pheochromocytoma/drug therapy , Actins/metabolism , Adrenal Gland Neoplasms/metabolism , Adrenal Gland Neoplasms/pathology , Animals , Antigens, CD34/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Chromogranin A/metabolism , Female , Immunohistochemistry , Isotretinoin/administration & dosage , Lovastatin/administration & dosage , Lung Neoplasms/metabolism , Lung Neoplasms/prevention & control , Lung Neoplasms/secondary , Mice , Mice, Nude , Muscle, Smooth/chemistry , Pheochromocytoma/metabolism , Pheochromocytoma/pathology , Time Factors , Transplantation, Homologous , Treatment Outcome , Tumor Burden/drug effects , Tyrosine 3-Monooxygenase/metabolism
19.
Am J Pathol ; 184(4): 953-965, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24518567

ABSTRACT

Medullary thyroid carcinoma is a relatively rare tumor with poor prognosis and therapy response. Its phenotype is determined by both genetic alterations (activating RET oncoprotein) and physiological stresses, namely hypoxia [activating hypoxia-inducible factor (HIF)]. Here, we investigated the cooperation between these two mechanisms. The idea emerged from the immunohistochemical analysis of carbonic anhydrases (CA) IX and XII expression in thyroid cancer. Although CAXII was present in all types of thyroid carcinomas, CAIX, a direct HIF target implicated in tumor progression, was associated with aggressive medullary and anaplastic carcinomas, and its expression pattern in medullary thyroid carcinomas suggested contribution of both hypoxic and oncogenic signaling. Therefore, we analyzed the CA9 promoter activity in transfected tumor cells expressing RET and/or the HIF-α subunit. We showed that overexpression of both wild-type and mutant RET can increase the CA9 promoter activity induced by HIF-1 (but not HIF-2) in hypoxia. Similar results were obtained with another HIF-1-regulated promoter derived from the lactate dehydrogenase A gene. Moreover, inhibition of the major kinase pathways, which transmit signals from RET and regulate HIF-1, abrogated their cooperative effect on the CA9 promoter. Thus, we brought the first experimental evidence for the crosstalk between RET and HIF-1 that can explain the increased expression of CAIX in medullary thyroid carcinoma and provide a rationale for therapy simultaneously targeting both pathways.


Subject(s)
Antigens, Neoplasm/metabolism , Carbonic Anhydrases/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Proto-Oncogene Proteins c-ret/metabolism , Signal Transduction , Thyroid Neoplasms/metabolism , Blotting, Western , Carbonic Anhydrase IX , Carcinoma, Neuroendocrine , Cell Line, Tumor , Humans , Immunohistochemistry , RNA Interference , Real-Time Polymerase Chain Reaction , Receptor Cross-Talk/physiology , Signal Transduction/physiology , Transfection
20.
Endocrinology ; 155(1): 27-32, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24189137

ABSTRACT

Pheochromocytomas (PHEOs) and paragangliomas (PGLs; extra-adrenal tumors) are rare neuroendocrine chromaffin cell tumors with a hereditary background in about 30%-35%. Those caused by succinate dehydrogenase subunit B (SDHB) germline mutations are associated with a high metastatic potential and ultimately higher patient mortality. Succinate dehydrogenase converts succinate to fumarate, uniquely linking the Krebs cycle and oxidative phosphorylation. SDH mutations result in the accumulation of succinate associated with various metabolic disturbances and the shift to aerobic glycolysis in tumor tissue. In the present study, we measured succinate and fumarate levels in mouse pheochromocytoma (MPC) and mouse tumor tissue (MTT) cells and in 10 apparently sporadic, 10 SDHB-, 5 SDHD-, and 2 neurofibromatosis 1-related PHEOs/PGLs and plasma samples using mass spectrometry. We found that the succinate-to-fumarate ratio was significantly higher in the SDHB- and SDHD-related PGLs than in apparently sporadic and neurofibromatosis 1-related PHEOs/PGLs (P = .0376). To further support our data, we silenced SDHB expression in MPC and MTT cells and evaluated the succinate and fumarate levels. Compared with control samples, SDHB-silenced MTT cells also showed an increase in the succinate-to-fumarate ratio (MTT cells: 2.45 vs 7.53), similar to the findings in SDHB-related PGLs. The present findings for the first time demonstrate a significantly increased succinate-to-fumarate ratio in SDHB/D-related PGLs and thus suggest this ratio may be used as a new metabolic marker for the detection of SDHB/D-related PHEOs/PGLs.


Subject(s)
Fumarates/chemistry , Gene Expression Regulation, Neoplastic , Paraganglioma/metabolism , Succinate Dehydrogenase/genetics , Succinic Acid/chemistry , Adrenal Gland Neoplasms/genetics , Adrenal Gland Neoplasms/metabolism , Animals , Biomarkers, Tumor/chemistry , Citric Acid Cycle , Female , Gene Expression Regulation, Enzymologic , Gene Silencing , Humans , Male , Mice , Mutation , Oxidative Phosphorylation , Phosphorylation
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