Young Suk Jo

Pharmacological Inhibition of Poly(ADP-Ribose) Polymerases Improves Fitness and Mitochondrial Function in Skeletal Muscle

We previously demonstrated that the deletion of the poly(ADP-ribose)polymerase (Parp)-1 gene in mice enhances oxidative metabolism, thereby protecting against diet-induced obesity. However, the therapeutic use of PARP inhibitors to enhance mitochondrial function remains to be explored. Here, we show tight negative correlation between Parp-1 expression and energy expenditure in heterogeneous mouse populations, indicating that variations in PARP-1 activity have an impact on metabolic homeostasis. Notably, these genetic correlations can be translated into pharmacological applications. Long-term treatment with PARP inhibitors enhances fitness in mice by increasing the abundance of mitochondrial respiratory complexes and boosting mitochondrial respiratory capacity. Furthermore, PARP inhibitors reverse mitochondrial defects in primary myotubes of obese humans and attenuate genetic defects of mitochondrial metabolism in human fibroblasts and C. elegans. Overall, our work validates in worm, mouse, and human models that PARP inhibition may be used to treat both genetic and acquired muscle dysfunction linked to defective mitochondrial function.

Pharmacological Stimulation of NADH Oxidation Ameliorates Obesity and Related Phenotypes in Mice

OBJECTIVE Nicotinamide adenine dinucleotides (NAD+ and NADH) play a crucial role in cellular energy metabolism, and a dysregulated NAD+-to-NADH ratio is implicated in metabolic syndrome. However, it is still unknown whether a modulating intracellular NAD+-to-NADH ratio is beneficial in treating metabolic syndrome. We tried to determine whether pharmacological stimulation of NADH oxidation provides therapeutic effects in rodent models of metabolic syndrome. RESEARCH DESIGN AND METHODS We used β-lapachone (βL), a natural substrate of NADH:quinone oxidoreductase 1 (NQO1), to stimulate NADH oxidation. The βL-induced pharmacological effect on cellular energy metabolism was evaluated in cells derived from NQO1-deficient mice. In vivo therapeutic effects of βL on metabolic syndrome were examined in diet-induced obesity (DIO) and ob/ob mice. RESULTS NQO1-dependent NADH oxidation by βL strongly provoked mitochondrial fatty acid oxidation in vitro and in vivo. These effects were accompanied by activation of AMP-activated protein kinase and carnitine palmitoyltransferase and suppression of acetyl-coenzyme A (CoA) carboxylase activity. Consistently, systemic βL administration in rodent models of metabolic syndrome dramatically ameliorated their key symptoms such as increased adiposity, glucose intolerance, dyslipidemia, and fatty liver. The treated mice also showed higher expressions of the genes related to mitochondrial energy metabolism (PPARγ coactivator-1α, nuclear respiratory factor-1) and caloric restriction (Sirt1) consistent with the increased mitochondrial biogenesis and energy expenditure. CONCLUSIONS Pharmacological activation of NADH oxidation by NQO1 resolves obesity and related phenotypes in mice, opening the possibility that it may provide the basis for a new therapy for the treatment of metabolic syndrome.

A SIRT7-dependent acetylation switch of GABPβ1 controls mitochondrial function

Mitochondrial activity is controlled by proteins encoded by both nuclear and mitochondrial DNA. Here, we identify Sirt7 as a crucial regulator of mitochondrial homeostasis. Sirt7 deficiency in mice induces multisystemic mitochondrial dysfunction, which is reflected by increased blood lactate levels, reduced exercise performance, cardiac dysfunction, hepatic microvesicular steatosis, and age-related hearing loss. This link between SIRT7 and mitochondrial function is translatable in humans, where SIRT7 overexpression rescues the mitochondrial functional defect in fibroblasts with a mutation in NDUFSI. These wide-ranging effects of SIRT7 on mitochondrial homeostasis are the consequence of the deacetylation of distinct lysine residues located in the hetero- and homodimerization domains of GABPβ1, a master regulator of nuclear-encoded mitochondrial genes. SIRT7-mediated deacetylation of GABPβ1 facilitates complex formation with GABPα and the transcriptional activation of the GABPα/GABPβ heterotetramer. Altogether, these data suggest that SIRT7 is a dynamic nuclear regulator of mitochondrial function through its impact on GABPβ1 function.

Diagnostic value of pyrosequencing for the BRAFV600E mutation in ultrasound-guided fine-needle aspiration biopsy samples of thyroid incidentalomas

Context  Dideoxy sequencing is the most commonly used method for detecting the BRAFV600E mutation in thyroid cancer and melanoma. However, this gold standard method often makes less definite results in detecting the BRAFV600E mutation when there are relatively low amounts of the mutant template in biopsy specimens, which are invariably contaminated with normal tissues. Pyrosequencing, which measures the incorporation of each of the four nucleotides at each template position and indicates the amounts of mutant template present, may be more useful in such situations.

Prediction of Occult Central Lymph Node Metastasis in Papillary Thyroid Carcinoma by Preoperative BRAF Analysis Using Fine-Needle Aspiration Biopsy: A Prospective Study

CONTEXT Few reports have determined whether preoperative detection of the BRAF V600E mutation in fine-needle aspiration biopsy (FNAB) may influence determination of surgical extent such as prophylactic central lymph node dissection (CLND) in patients with papillary thyroid carcinoma (PTC). OBJECTIVES Our objectives were to investigate whether preoperative BRAF analysis may assist determination of surgical extent, including prophylactic CLND with variable clinicopathological risk factors for central lymph node metastasis, in patients with PTC and clinically node-negative neck. PATIENTS AND METHODS From July 2009 to May 2011, we prospectively enrolled 148 PTC patients with clinically node-negative neck who received a total thyroidectomy and prophylactic CLND. BRAF mutation by pyrosequencing was tested on preoperative FNAB specimens. The relationships between occult central lymph node metastasis and preoperative BRAF mutation or clinicopathological factors were analyzed. Additionally, we assessed the associations between preoperative BRAF mutation status and various clinicopathological characteristics of PTC revealed postoperatively. RESULTS The prevalence of the BRAF V600E mutation was 53.4%, and the rate of occult central lymph node metastasis was 25.7%. Multivariate analysis showed that tumor size over 1 cm [P = 0.006; odds ratio (OR) = 3.559], perithyroidal invasion (P = 0.023; OR = 2.893), and preoperative positive BRAF mutation (P = 0.029; OR = 2.727) were independent risk factors for the presence of occult central lymph node metastasis. BRAF mutation examined in FNAB specimens, compared with the wild-type allele, strongly predicted perithyroidal invasion (48 vs. 29%; P = 0.017), extracapsular spread (65 vs. 45%; P = 0.017), occult central lymph node metastasis (35 vs. 15%; P = 0.004), and advanced TNM stage (44 vs. 28%; P = 0.035). In the multivariate analysis, patients with preoperative positive BRAF mutation were significantly more likely (P = 0.023; OR = 2.848) to have occult central lymph node metastasis. CONCLUSION Preoperative BRAF analysis by FNAB and primary tumor size based on ultrasonography may assist in predicting occult central lymph node metastasis in patients with PTC and clinically node-negative neck.

Crif1 Deficiency Reduces Adipose OXPHOS Capacity and Triggers Inflammation and Insulin Resistance in Mice

Impaired mitochondrial oxidative phosphorylation (OXPHOS) has been proposed as an etiological mechanism underlying insulin resistance. However, the initiating organ of OXPHOS dysfunction during the development of systemic insulin resistance has yet to be identified. To determine whether adipose OXPHOS deficiency plays an etiological role in systemic insulin resistance, the metabolic phenotype of mice with OXPHOS–deficient adipose tissue was examined. Crif1 is a protein required for the intramitochondrial production of mtDNA–encoded OXPHOS subunits; therefore, Crif1 haploinsufficient deficiency in mice results in a mild, but specific, failure of OXPHOS capacity in vivo. Although adipose-specific Crif1-haploinsufficient mice showed normal growth and development, they became insulin-resistant. Crif1-silenced adipocytes showed higher expression of chemokines, the expression of which is dependent upon stress kinases and antioxidant. Accordingly, examination of adipose tissue from Crif1-haploinsufficient mice revealed increased secretion of MCP1 and TNFα, as well as marked infiltration by macrophages. These findings indicate that the OXPHOS status of adipose tissue determines its metabolic and inflammatory responses, and may cause systemic inflammation and insulin resistance.

Mitochondrial Localization and Regulation of BRAFV600E in Thyroid Cancer: A Clinically Used RAF Inhibitor Is Unable to Block the Mitochondrial Activities of BRAFV600E

CONTEXT The oncogenic BRAF(V600E) mutation results in an active structural conformation characterized by greatly elevated ERK activity. However, additional cellular effects caused by subcellular action of BRAF(V600E) remain to be identified. OBJECTIVE To explore these effects, differences in the subcellular localization of wild-type and mutant BRAF in thyroid cancer were investigated. RESULTS A significant proportion of endogenous and exogenous BRAF(V600E), but not wild-type BRAF, was detected in the mitochondrial fraction, similar to other BRAF mutants including BRAF(V600D), BRAF(V600K), BRAF(V600R), and BRAF(G469A), which showed elevated kinase activity and mitochondrial localization. Induced expression of BRAF(V600E) suppressed the apoptotic responses against staurosporine and TNFα/cycloheximide. Interestingly, the mitochondrial localization and antiapoptotic activities of BRAF(V600E) were unaffected by sorafenib and U0126 suppression of MAPK kinase (MEK) and ERK activities. Similarly, although the RAF inhibitor sorafenib effectively inhibited MEK/ERK activation, it did not block the mitochondrial localization of BRAF(V600E). In addition, inducible expression of BRAF(V600E) increased the glucose uptake rate and decreased O(2) consumption, suggesting that BRAF(V600E) reduces mitochondrial oxidative phosphorylation, a signature feature of cancer cells. Again, these metabolic alterations resulted by BRAF(V600E) expression were not affected by the treatment of thyroid cells by sorafenib. Therefore, RAF and MEK inhibitors are unable to block the antiapoptotic activity of BRAF(V600E) or correct the high glucose uptake rate and glycolytic activity and suppressed mitochondrial oxidative phosphorylation induced by BRAF(V600E). CONCLUSIONS The mitochondrial localization observed in oncogenic BRAF mutants might be related to their altered responses to apoptotic stimuli and characteristic metabolic phenotypes found in thyroid cancer. The inability of MEK and RAF inhibitors, U0126 and sorafenib, respectively, to block the mitochondrial localization of BRAF(V600E) has additional therapeutic implications for BRAF(V600E)-positive thyroid cancers.

L1 Cell Adhesion Molecule Is a Novel Independent Poor Prognostic Factor of Extrahepatic Cholangiocarcinoma

Purpose: Cholangiocarcinomas (CC) are associated with poor survival, but diagnostic markers and therapeutic targets have not yet been elucidated. We previously found aberrant expression of L1 cell adhesion molecule in intrahepatic CC and a role for L1 in the progression of intrahepatic CC. Here, we analyzed L1 expression in extrahepatic CC (ECC) and evaluated its prognostic significance. Experimental Design: We examined L1 expression in tumors from 75 ECC patients by immunohistochemistry. We analyzed the correlations between L1 expression and clinicopathologic factors as well as patient survival. Results: L1 was not expressed in normal extrahepatic bile duct epithelium but was aberrantly expressed in 42.7% of ECC tumors. High expression of L1 was detected at the invasive front of tumors and was significantly associated with perineural invasion (P < 0.01). Univariate analysis indicated that various prognostic factors such as histologic grade 3, advanced pathologic T stage and clinical stage, perineural invasion, nodal metastasis, and high expression of L1 were risk factors predicting patient survival. Multivariate analyses done by Coxs proportional hazards model showed that high expression of L1 (hazard ratio, 2.171; 95% confidence interval, 1.162-4.055; P = 0.015) and nodal metastasis (hazard ratio, 2.088; 95% confidence interval, 1.159-3.764; P = 0.014) were independent risk factors for patient death. Conclusions: L1 was highly expressed in 42.7% of ECC and its expression was significantly associated with perineural invasion. High expression of L1 and nodal metastasis were independent poor prognostic factors predicting overall survival in patients with ECC. (Clin Cancer Res 2009;15(23):7345–51)

Pyrosequencing cut‐off value identifying BRAFV600E mutation in fine needle aspiration samples of thyroid nodules

Context  Recently, tremendous efforts have been made towards the development of sensitive techniques to detect the BRAFV600E mutation in fine needle aspiration biopsy (FNAB) samples. However, newly developed quantitative and semi‐quantitative methods, such as dual‐priming oligonucleotide (DPO)‐based multiplex polymerase chain reaction (PCR), have the potential to generate false‐positive (FP) results.

Cross-Regulation between Oncogenic BRAFV600E Kinase and the MST1 Pathway in Papillary Thyroid Carcinoma

Background The BRAFV600E mutation leading to constitutive signaling of MEK-ERK pathways causes papillary thyroid cancer (PTC). Ras association domain family 1A (RASSF1A), which is an important regulator of MST1 tumor suppressor pathways, is inactivated by hypermethylation of its promoter region in 20 to 32% of PTC. However, in PTC without RASSF1A methylation, the regulatory mechanisms of RASSF1A-MST1 pathways remain to be elucidated, and the functional cooperation or cross regulation between BRAFV600E and MST1,which activates Foxo3,has not been investigated. Methodology/Principal Findings The negative regulators of the cell cycle, p21 and p27, are strongly induced by transcriptional activation of FoxO3 in BRAFV600E positive thyroid cancer cells. The FoxO3 transactivation is augmented by RASSF1A and the MST1 signaling pathway. Interestingly, introduction of BRAFV600Emarkedly abolished FoxO3 transactivation and resulted in the suppression of p21 and p27 expression. The suppression of FoxO3 transactivation by BRAFV600Eis strongly increased by coexpression of MST1 but it is not observed in the cells in which MST1, but not MST2,is silenced. Mechanistically, BRAFV600Ewas able to bind to the C-terminal region of MST1 and resulted in the suppression of MST1 kinase activities. The induction of the G1-checkpoint CDK inhibitors, p21 and p27,by the RASSF1A-MST1-FoxO3 pathway facilitates cellular apoptosis, whereasaddition of BRAFV600E inhibits the apoptotic processes through the inactivation of MST1. Transgenic induction of BRAFV600Ein the thyroid gland results in cancers resembling human papillary thyroid cancers. The development of BRAFV600Etransgenic mice with the MST1 knockout background showed that these mice had abundant foci of poorly differentiated carcinomas and large areas without follicular architecture or colloid formation. Conclusions/Significance The results of this study revealed that the oncogenic effect of BRAFV600E is associated with the inhibition of MST1 tumor suppressor pathways, and that the activity of RASSF1A-MST1-FoxO3 pathways determines the phenotypes of BRAFV600E tumors.