Anti-Cancer Potential of MAPK Pathway Inhibition in Paragangliomas–Effect of Different Statins on Mouse Pheochromocytoma Cells

To date, malignant pheochromocytomas and paragangliomas (PHEOs/PGLs) cannot be effectively cured and thus novel treatment strategies are urgently needed. Lovastatin has been shown to effectively induce apoptosis in mouse PHEO cells (MPC) and the more aggressive mouse tumor tissue-derived cells (MTT), which was accompanied by decreased phosphorylation of mitogen-activated kinase (MAPK) pathway players. The MAPK pathway plays a role in numerous aggressive tumors and has been associated with a subgroup of PHEOs/PGLs, including K-RAS-, RET-, and NF1-mutated tumors. Our aim was to establish whether MAPK signaling may also play a role in aggressive, succinate dehydrogenase (SDH) B mutation-derived PHEOs/PGLs. Expression profiling and western blot analysis indicated that specific aspects of MAPK-signaling are active in SDHB PHEOs/PGLs, suggesting that inhibition by statin treatment could be beneficial. Moreover, we aimed to assess whether the anti-proliferative effect of lovastatin on MPC and MTT differed from that exerted by fluvastatin, simvastatin, atorvastatin, pravastatin, or rosuvastatin. Simvastatin and fluvastatin decreased cell proliferation most effectively and the more aggressive MTT cells appeared more sensitive in this respect. Inhibition of MAPK1 and 3 phosphorylation following treatment with fluvastatin, simvastatin, and lovastatin was confirmed by western blot. Increased levels of CASP-3 and PARP cleavage confirmed induction of apoptosis following the treatment. At a concentration low enough not to affect cell proliferation, spontaneous migration of MPC and MTT was significantly inhibited within 24 hours of treatment. In conclusion, lipophilic statins may present a promising therapeutic option for treatment of aggressive human paragangliomas by inducing apoptosis and inhibiting tumor spread.     

Ribonucleoprotein Y-box-binding protein-1 regulates mitochondrial oxidative phosphorylation (OXPHOS) protein expression after serum stimulation through binding to OXPHOS mRNA

Mitochondria play key roles in essential cellular functions, such as energy production, metabolic pathways and aging. Growth factor-mediated expression of the mitochondrial OXPHOS (oxidative phosphorylation) complex proteins has been proposed to play a fundamental role in metabolic homoeostasis. Although protein translation is affected by general RNA-binding proteins, very little is known about the mechanism involved in mitochondrial OXPHOS protein translation. In the present study, serum stimulation induced nuclear-encoded OXPHOS protein expression, such as NDUFA9 [NADH dehydrogenase (ubiquinone) 1α subcomplex, 9, 39 kDa], NDUFB8 [NADH dehydrogenase (ubiquinone) 1β subcomplex, 8, 19 kDa], SDHB [succinate dehydrogenase complex, subunit B, iron sulfur (Ip)] and UQCRFS1 (ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1), and mitochondrial ATP production, in a translation-dependent manner. We also observed that the major ribonucleoprotein YB-1 (Y-box-binding protein-1) preferentially bound to these OXPHOS mRNAs and regulated the recruitment of mRNAs from inactive mRNPs (messenger ribonucleoprotein particles) to active polysomes. YB-1 depletion led to up-regulation of mitochondrial function through induction of OXPHOS protein translation from inactive mRNP release. In contrast, YB-1 overexpression suppressed the translation of these OXPHOS mRNAs through reduced polysome formation, suggesting that YB-1 regulated the translation of mitochondrial OXPHOS mRNAs through mRNA binding. Taken together, our findings suggest that YB-1 is a critical factor for translation that may control OXPHOS activity.     

Chromogranin A in a Cohort of Pheochromocytomas and Paragangliomas: Usefulness at Diagnosis and as an Early Biomarker of Recurrence

ObjectiveTo evaluate the usefulness of chromogranin A (CgA) in the management of patients with pheochromocytomas (PHEOs) and paragangliomas (PGLs).MethodsWe retrospectively reviewed the charts of 132 patients with confirmed PHEOs/PGLs (PPGLs) followed at our medical center. CgA was measured in 80 patients at diagnosis. The exclusion criteria removed 19 of these patients. Five patients with relapses were also analyzed.ResultsOur cohort of 61 patients included 34 PHEOs, 14 head and neck PGLs, and 13 thoracoabdominal PGLs. CgA levels were elevated in 53 of 61 patients (86.9%) at diagnosis: 33 of 34 (97.1%) PHEOs, 9 of 14 (64.3%) head and neck paragangliomas, and 11 of 13 (84.6%) thoracoabdominal paragangliomas. For 8 of 13 (61.5%) nonfunctional PPGLs (5 head and neck paragangliomas and 3 thoracoabdominal paragangliomas), increased CgA levels showed potential as a tumor marker during follow-up. Of 10 patients with malignant PPGLs, only 1 had normal CgA levels (10.0%). Among 54 patients with PPGLs who underwent genetic testing, elevated CgA levels were positive in 73.7% of patients carrying a germline genetic variant (pathogenic and of unknown significance) versus 91.4% of patients without a known germline variant. We also report 5 PPGL cases with increased CgA levels as the first detectable marker of tumoral recurrence or progression preceding other biochemical markers or imaging.ConclusionCgA is a sensitive marker for the diagnosis of PHEO (97.1%) and thoracoabdominal paraganglioma (84.6%). CgA may be useful in the follow-up of nonfunctional PGLs and may also play a complementary role in the early detection of recurrence in secreting PPGLs.     

Actuality of Warburg’s views in our understanding of renal cancer metabolism

More than 50 years ago, Warburg proposed that the shift in glucose metabolism from oxidative phosphorylation (OXPHOS) to glycolysis occurring in spite of an adequate oxygen supply was at the root of cancer. This hypothesis often disregarded over the following years has recently stirred up much interest due to progress made in cancer genetics and proteomics. Studies related to renal cancers have been particularly informative to understand how abnormal use of glucose and decrease in OXPHOS are linked to cell proliferation in tumors. Indeed, in aggressive tumors such as clear cell renal carcinoma, the von Hippel–Lindau factor invalidation stabilizes the hypoxia-inducible factor (HIF) in the presence of oxygen. HIF stimulating glycolytic gene expression increases the glycolytic flux. Deficiencies in genes involved in oxidative phosphorylation that can explain the down-regulation of OXPHOS components also begin to be identified. These findings are important in the search for novel therapeutic approaches to cancer treatment.     

肿瘤能量代谢：糖酵解还是氧化磷酸化？

正常细胞代谢活动所需要的能量主要由线粒体氧化磷酸化产生的ATP提供。与正常细胞不同,肿瘤细胞糖酵解增强,氧化磷酸化功能降低。长期以来,肿瘤细胞的有氧糖酵解被认为是由于线粒体出现不可逆的损伤。最近有不少研究结果对这一观点提出质疑,认为多数肿瘤的线粒体氧化磷酸化功能是完好的,肿瘤有氧糖酵解的改变被认为是其他多种因素（例如癌基因、肿瘤抑制基因、低氧微环境、mtDNA突变等）综合作用的结果。     

Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells

Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells. oxygen consumption; oxidative phosphorylation; Warburg effect; real time     

Mitochondrial metabolism in cancer metastasis: Visualizing tumor cell mitochondria and the “reverse Warburg effect” in positive lymph node tissue

We have recently proposed a new two-compartment model for understanding the Warburg effect in tumor metabolism. In this model, glycolytic stromal cells produce mitochondrial fuels (L-lactate and ketone bodies) that are then transferred to oxidative epithelial cancer cells, driving OXPHOS and mitochondrial metabolism. Thus, stromal catabolism fuels anabolic tumor growth via energy transfer. We have termed this new cancer paradigm the “reverse Warburg effect,” because stromal cells undergo aerobic glycolysis, rather than tumor cells. To assess whether this mechanism also applies during cancer cell metastasis, we analyzed the bioenergetic status of breast cancer lymph node metastases, by employing a series of metabolic protein markers. For this purpose, we used MCT4 to identify glycolytic cells. Similarly, we used TOMM20 and COX staining as markers of mitochondrial mass and OXPHOS activity, respectively. Consistent with the “reverse Warburg effect,” our results indicate that metastatic breast cancer cells amplify oxidative mitochondrial metabolism (OXPHOS) and that adjacent stromal cells are glycolytic and lack detectable mitochondria. Glycolytic stromal cells included cancer-associated fibroblasts, adipocytes and inflammatory cells. Double labeling experiments with glycolytic (MCT4) and oxidative (TOMM20 or COX) markers directly shows that at least two different metabolic compartments co-exist, side-by-side, within primary tumors and their metastases. Since cancer-associated immune cells appeared glycolytic, this observation may also explain how inflammation literally “fuels” tumor progression and metastatic dissemination, by “feeding” mitochondrial metabolism in cancer cells. Finally, MCT4(+) and TOMM20(-) “glycolytic” cancer cells were rarely observed, indicating that the conventional “Warburg effect” does not frequently occur in cancer-positive lymph node metastases.Key words: caveolin-1, oxidative stress, MCT4, metabolic coupling, tumor stroma, SLC16A3, monocarboxylic acid transporter, two-compartment tumor metabolism, metastasis, TOMM20, complex IV, OXPHOS, mitochondria, inflammation     

Case Report: Genetic Alterations Associated with the Progression of Carotid Paraganglioma

Paragangliomas (PGLs) are rare neuroendocrine tumors that can develop from any paraganglion across the body. The carotid body is the most often location of PGLs in the head and neck region. Carotid PGLs (CPGLs) are characterized by predominantly non-aggressive behavior; however, all tumors have the potential to metastasize. To date, molecular mechanisms of paraganglioma progression remain elusive. We report a case of a 38-year-old woman with metastatic CPGL manifesting as a recurrent tumor with lymph node metastasis. The tumor was fast-growing and had a high Ki-67 proliferation index. Immunohistochemical (IHC) examination and whole-exome sequencing were performed for both recurrent tumor and metastasis. A germline pathogenic splice acceptor variant in the SDHB gene was found in the patient. Immunoreactivity of the SDHB subunit was weak diffuse in both samples, indicating deficiency of the succinate dehydrogenase. Moreover, the recurrent tumor exhibited loss of heterozygosity (LOH) at the SDHB locus, that is according to Knudson’s "two-hit" hypothesis of cancer causation. We also identified a rare somatic promotor mutation in the TERT gene associated with the tumor progression. Obtained results confirmed the indicative role of the germline SDHB mutation for metastatic CPGLs, as well as the potential prognostic value of the TERT promoter mutation.     

Negative modulation of mitochondrial oxidative phosphorylation by epigallocatechin-3 gallate leads to growth arrest and apoptosis in human malignant pleural mesothelioma cells

Increasing evidence reveals a large dependency of epithelial cancer cells on oxidative phosphorylation (OXPHOS) for energy production. In this study we tested the potential of epigallocatechin-3-gallate (EGCG), a natural polyphenol known to target mitochondria, in inducing OXPHOS impairment and cell energy deficit in human epitheliod (REN cells) and biphasic (MSTO-211H cells) malignant pleural mesothelioma (MMe), a rare but highly aggressive tumor with high unmet need for treatment. Due to EGCG instability that causes H₂O₂ formation in culture medium, the drug was added to MMe cells in the presence of exogenous superoxide dismutase and catalase, already proved to stabilize the EGCG molecule and prevent EGCG-dependent reactive oxygen species formation. We show that under these experimental conditions, EGCG causes the selective arrest of MMe cell growth with respect to normal mesothelial cells and the induction of mitochondria-mediated apoptosis, as revealed by early mitochondrial ultrastructure modification, swelling and cytochrome c release. We disclose a novel mechanism by which EGCG induces apoptosis through the impairment of mitochondrial respiratory chain complexes, particularly of complex I, II and ATP synthase. This induces a strong reduction in ATP production by OXPHOS, that is not adequately counterbalanced by glycolytic shift, resulting in cell energy deficit, cell cycle arrest and apoptosis. The EGCG-dependent negative modulation of mitochondrial energy metabolism, selective for cancer cells, gives an important input for the development of novel pharmacological strategies for MMe.     

Mitochondrial metabolism in cancer metastasis

We have recently proposed a new two-compartment model for understanding the Warburg effect in tumor metabolism. In this model, glycolytic stromal cells produce mitochondrial fuels (L-lactate and ketone bodies) that are then transferred to oxidative epithelial cancer cells, driving OXPHOS and mitochondrial metabolism. Thus, stromal catabolism fuels anabolic tumor growth via energy transfer. We have termed this new cancer paradigm the “reverse Warburg effect,” because stromal cells undergo aerobic glycolysis, rather than tumor cells. To assess whether this mechanism also applies during cancer cell metastasis, we analyzed the bioenergetic status of breast cancer lymph node metastases, by employing a series of metabolic protein markers. For this purpose, we used MCT4 to identify glycolytic cells. Similarly, we used TO MM20 and COX staining as markers of mitochondrial mass and OXPHOS activity, respectively. Consistent with the “reverse Warburg effect,” our results indicate that metastatic breast cancer cells amplify oxidative mitochondrial metabolism (OXPHOS) and that adjacent stromal cells are glycolytic and lack detectable mitochondria. Glycolytic stromal cells included cancer-associated fibroblasts, adipocytes and inflammatory cells. Double labeling experiments with glycolytic (MCT4) and oxidative (TO MM20 or COX) markers directly shows that at least two different metabolic compartments co-exist, side-by-side, within primary tumors and their metastases. Since cancer-associated immune cells appeared glycolytic, this observation may also explain how inflammation literally “fuels” tumor progression and metastatic dissemination, by “feeding” mitochondrial metabolism in cancer cells. Finally, MCT4(+) and TO MM20(-) “glycolytic” cancer cells were rarely observed, indicating that the conventional “Warburg effect” does not frequently occur in cancer-positive lymph node metastases.     

Peptide receptor radionuclide therapy (PRRT) with 177Lu-DOTATATE in individuals with neck or mediastinal paraganglioma (PGL)

Paragangliomas (PGLs) are neuroendocrine tum-ors that arise embryologically from the neural crest. Sympathetic PGLs can be located in the thoracic-abdominal region while parasympathetic PGLs are mainly situated in the head and neck region. Most PGLs are sporadic, but in 30% of cases they are hereditary (associated with mutations of SDHB, SDHC, SDHD, SDHAF2, SDHA, TMEM, MAX, and VHL); they can be classified into 4 different paraganglioma syndromes: PGL1, PGL2, PGL3, and PGL4. Surgery is the treatment of choice for both sympathetic and parasympathetic PGLs. Other types of treatment include medical agents (such as gemcitabine, cisplatin, or sunitinib) and radiotherapy (external-beam radiotherapy or stereotactic surgery). Surgery and radiotherapy, however, can cause important side effects such as vascular complications and peripheral nerve damage (hypoglossal, recurrent laryngeal, glossopharyngeal, and vagus). Another possible treatment option is the use of peptide receptor radionuclide therapy (PRRT), including PRRT with 177Lu-DOTATATE. We studied 4 patients with hereditary nonmetastatic paraganglioma syndrome type 1 (PGL1), with progressive disease, in whom surgical excision was not possible. They were treated with 177Lu-DOTATATE (3-5 cycles) and all had a partial response (PR) or a stable disease (SD) to the treatment. In conclusion, a good alternative treatment when surgical or radiation therapy are contraindicated could be radiometabolic therapy with 177Lu-DOTATATE.     

AIF deficiency compromises oxidative phosphorylation

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, after apoptosis induction, translocates to the nucleus where it participates in apoptotic chromatinolysis. Here, we show that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity. Although AIF itself is not a part of complex I, AIF-deficient cells exhibit a reduced content of complex I and of its components, pointing to a role of AIF in the biogenesis and/or maintenance of this polyprotein complex. Harlequin mice with reduced AIF expression due to a retroviral insertion into the AIF gene also manifest a reduced oxidative phosphorylation (OXPHOS) in the retina and in the brain, correlating with reduced expression of complex I subunits, retinal degeneration, and neuronal defects. Altogether, these data point to a role of AIF in OXPHOS and emphasize the dual role of AIF in life and death.     

Retinoblastoma protein promotes oxidative phosphorylation through upregulation of glycolytic genes in oncogene‐induced senescent cells

Metabolism is closely linked with cellular state and biological processes, but the mechanisms controlling metabolic properties in different contexts remain unclear. Cellular senescence is an irreversible growth arrest induced by various stresses, which exhibits active secretory and metabolic phenotypes. Here, we show that retinoblastoma protein (RB) plays a critical role in promoting the metabolic flow by activating both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) in cells that have undergone oncogene‐induced senescence (OIS). A combination of real‐time metabolic monitoring, and metabolome and gene expression analyses showed that OIS‐induced fibroblasts developed an accelerated metabolic flow. The loss of RB downregulated a series of glycolytic genes and simultaneously reduced metabolites produced from the glycolytic pathway, indicating that RB upregulates glycolytic genes in OIS cells. Importantly, both mitochondrial OXPHOS and glycolytic activities were abolished in RB‐depleted or downstream glycolytic enzyme‐depleted OIS cells, suggesting that RB‐mediated glycolytic activation induces a metabolic flux into the OXPHOS pathway. Collectively, our findings reveal that RB essentially functions in metabolic remodeling and the maintenance of the active energy production in OIS cells.     

Characteristics And Outcomes Of Metastatic Sdhb And Sporadic Pheochromocytoma/Paraganglioma: An National Institutes Of Health Study

Objective: Overall about 10 to 20% of pheochromocytomas/paragangliomas (PHEOs/PGLs) are metastatic, with higher metastatic potential observed in succinate dehydrogenase subunit B/fumarate hydratase (SDHB/FH)-related tumors. Due to the improved availability of biochemical and genetic testing and the frequent use of anatomical/functional imaging, there is currently a higher detection rate of metastatic PHEO/PGL.Methods: A retrospective analysis of 132 patients (27 children, 105 adults) with metastatic PHEO/PGL diagnosed and treated from 2000 to 2014 was conducted.Results: Seventy-seven (58%) males and 55 (42%) females were included; 39 (30%) have died, with no sex preference. Seventy-three (55%) patients had SDHB mutations; 59 (45%) patients had apparently sporadic tumors (AST). SDHB patients had an average age at primary tumor diagnosis of 31 ± 16 years compared to 40 ± 15 years in AST patients (P<.001). The average metastatic interval (MI) decreased with increasing age in both SDHB and AST patients (P = .013 for both). Only 16% of all primary tumors were smaller than 4.5 cm. Eleven percent of patients had biochemically silent disease, more with SDHB. Of SDHB patients, 23% had metastatic tumors at first diagnosis, compared to 15% of AST patients. Five- and 10-year survival rates were significantly better for metastatic AST than SDHB patients (P = .01). Overall survival was significantly different between children and adults (P = .037); this was mostly attributed to the SDHB patients, in whom children had statistically significantly longer survival than adults (P = .006). The deceased patients all died due to the PHEO/PGL and mainly had noradrenergic phenotypes.Conclusion: In children, metastatic PHEOs/PGLs are mainly due to SDHB mutations; in adults they are equally distributed between in SDHB mutations and AST, with better 5- and 10-year survival rates for ASTs. In SDHB patients, children survive longer than adults. Primary metastatic tumors, most presenting as noradrenergic PGLs, are larger than 4.5 cm in >80% of patients. The frequency of metastatic tumors from primary AST increases with age, including a decreased MI compared to SDHB tumors. These results support several recommendations that are summarized in the Discussion.Abbreviations:A = adrenalAMTD = age at the initial metastatic tumor diagnosisAPTD = age of patients at the time of the primary tumor diagnosisAST = apparently sporadic tumorCI = confidence intervalCT = computed tomographyDA = dopamineEA = extra-adrenalEPI = epinephrine[18F]-FDA = [18F]-fluorodopamine[18F]-FDG = [18F]-fluorodeoxyglucoseFH = fumarate hydrataseHIF2A = hypoxia-inducible factor 2αMAX = myc-associated factor XMI = metastatic intervalMIBG = metaiodobenzylguanidineMN = metanephrineMRI = magnetic resonance imagingNE = norepinephrineNF1 = neurofibromatosis type 1NIH = National Institutes of HealthNMN = normetanephrinePET = positron emission tomographyPGL = paragangliomaPHEO = pheochromocytomaRET = rearranged during transfectionSDHA = succinate dehydrogenase subunit ASDHAF2 = encoding SDH complex assembly factor 2SDHB = succinate dehydrogenase subunit BSDHC = succinate dehydrogenase subunit CSDHD = succinate dehydrogenase subunit DTMEM127 = transmembrane protein 127VHL = von Hippel-Lindau