Collagen XI Alpha 1 (COL11A1) Expression in the Tumor Microenvironment Drives Neuroblastoma Dissemination.

BACKGROUND: Neuroblastoma (NB) is among the most common cancers in children. A highly aggressive form of cancer, NB relies on cells in the microenvironment for dissemination particularly cancer associated fibroblast (CAFs). CAFs synthesise the extracellular matrix to create a scaffold for tumor growth thus enabling the carcinogenesis of NB, Collagen, an abundant scaffold protein produced by CAFs, has been implicated in the creation of an optimal tumor microenvironment, however, the expression profile of collagen within NB is not yet known. METHODS: We characterised collagen expression within the tumor-stroma boundary by microarray and confirmed by qRT-PCR and immunohistochemistry. RESULTS: The collagen marker, COL11A1, was also upregulated in NB CD45+ cells and SMA+ CAFs. Furthermore, SMA+ CAFs led to neuroblastoma cell invasion in an in vitro co-culture system which was subsequently attenuated by gene silencing COL11A1. Immunohistochemical staining of clinical tumor samples revealed that high COL11A1 expression in the stroma adjacent to tumour site, significantly associated with advanced cancer stages, age ≥18 months, undifferentiated tumor status, relapse and poor overall survival. CONCLUSION: Collectively, these results suggest that a COL11A1 signature in the NB microenvironment could represent a novel target for therapeutic intervention.

Novel anti-adipogenic effect of CF3-allylated indole in 3T3-L1 cells.

Indole derivatives from various plants are known to have health benefits because of their anti-cancer, anti-oxidant, anti-inflammatory, and anti-tubercular effects. However, their effects on adipogenesis have not been fully elucidated yet. Herein, we show that a newly synthesized indole derivative, CF3-allylated indole, [(E)-1-(pyrimidin- 2-yl)-2-(4,4,4- trifluorobut-2-enyl)-1H-indole], effectively inhibits adipogenesis. We found that CF3-allylated indole inhibited lipid accumulation and suppressed the expression of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator activated receptor γ (PPARγ) in 3T3-L1 cells. The inhibitory effect of CF3-allylated indole primarily occurred at the early phase of adipocyte differentiation by increasing intracellular cyclic adenosine monophosphate (cAMP) levels and enhancing protein kinase A (PKA) and adenosine monophosphate-activated protein kinase (AMPK) signaling. Conversely, depletion of PKA or treatment with a protein kinase A inhibitor (H89) reversed such inhibitory effects of CF3-allylated indole on adipogenesis and PPARγ expression. These results suggest that CF3-allylated indole inhibits early stages of adipogenesis by increasing phosphorylation of PKA/AMPK, leading to decreased expression of adipogenic genes in 3T3-L1 cells. These results indicate that CF3-allylated indole has potential for controlling initial adipocyte differentiation in metabolic disorders such as obesity.

A novel nonsense mutation of ERCC2 in a Vietnamese family with xeroderma pigmentosum syndrome group D.

Xeroderma pigmentosum (XP) group D, a severe disease often typified by extreme sun sensitivity, can be caused by ERCC2 mutations. ERCC2 encodes an adenosine triphosphate (ATP)-dependent DNA helicase, namely XP group D protein (XPD). The XPD, one of ten subunits of the transcription factor TFIIH, plays a critical role in the nucleotide-excision repair (NER) pathway. Mutations in XPD that affect the NER pathway can lead to neurological degeneration and skin cancer, which are the most common causes of death in XP patients. Here, we present detailed phenotypic information on a Vietnamese family in which four members were affected by XP with extreme sun sensitivity. Genomic analysis revealed a compound heterozygous mutation of ERCC2 that affected family members and single heterozygous mutations in unaffected family members. We identified a novel, nonsense mutation in one allele of ERCC2 (c.1354C > T, p.Q452X) and a known missense mutation in the other allele (c.2048G > A, p.R683Q). Fibroblasts isolated from the compound heterozygous subject also failed to recover from UV-driven DNA damage, thus recapitulating aspects of XP syndrome in vitro. We describe a novel ERCC2 variant that leads to the breakdown of the NER pathway across generations of a family presenting with severe XP.

Carbamazepine Enhances Adipogenesis by Inhibiting Wnt/ß-catenin Expression.

Carbamazepine is a drug that is widely used in the treatment of epilepsy and bipolar disorder. The prevalence of obesity in patients treated with carbamazepine has been frequently reported. However, whether carbamazepine affects adipogenesis, one of the critical steps in the development of obesity, remains unclear. Here, we show that carbamazepine increased the expression levels of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein ß (C/EBPß), and fatty acid synthase (FASN) in 3T3-L1 cells. Notably, carbamazepine inhibited the expression levels of ß-catenin, a negative regulator of adipogenesis, leading to enhanced adipogenesis. Conversely, ß-catenin overexpression abolished the effect of carbamazepine on adipogenic gene expression. However, depletion of ß-catenin further enhanced PPARγ expression. In addition, carbamazepine reduced ß-catenin expression by lowering the levels of phospho-low density lipoprotein receptor-related protein 6 (p-LRP6) and phospho-glycogen synthase kinase 3ß (p-GSK3ß) in Wnt/ß-catenin signaling. Moreover, carbamazepine reduced Wnt mRNA expression and decreased the promoter activities of TCF, the target of ß-catenin during adipogenesis. These results suggest that carbamazepine enhances adipogenesis by suppressing Wnt/ß-catenin expression, indicating its potential effects on obesity-related metabolism.

Vacuolar H+-ATPase Subunit V0C Regulates Aerobic Glycolysis of Esophageal Cancer Cells via PKM2 Signaling.

The vacuolar H+-adenosine triphosphatase (ATPase) subunit V0C (ATP6V0C), a proton-conducting, pore-forming subunit of vacuolar ATPase, maintains pH homeostasis and induces organelle acidification. The intracellular and extracellular pH of cancer cells affects their growth; however, the role of ATP6V0C in highly invasive esophageal cancer cells (ECCs) remains unclear. In this study, we examined the role of ATP6V0C in glucose metabolism in ECCs. The ATP6V0C depletion attenuated ECC proliferation, invasion, and suppressed glucose metabolism, as indicated by reduced glucose uptake and decreased lactate and adenosine triphosphate (ATP) production in cells. Consistent with this, expression of glycolytic enzyme and the extracellular acidification rate (ECAR) were also decreased by ATP6V0C knockdown. Mechanistically, ATP6V0C interacted with pyruvate kinase isoform M2 (PKM2), a key regulator of glycolysis in ECCs. The ATP6V0C depletion reduced PKM2 phosphorylation at tyrosine residue 105 (Tyr105), leading to inhibition of nuclear translocation of PKM2. In addition, ATP6V0C was recruited at hypoxia response element (HRE) sites in the lactate dehydrogenase A (LDHA) gene for glycolysis. Thus, our data suggest that ATP6V0C enhances aerobic glycolysis and motility in ECCs.

A single extra copy of Down syndrome critical region 1-4 results in impaired hepatic glucose homeostasis.

OBJECTIVES: During fasting, hepatic gluconeogenesis is induced to maintain energy homeostasis. Moreover, abnormal dysregulation of hepatic glucose production is commonly observed in type 2 diabetes. However, the signaling components controlling hepatic glucose production to maintain normal glucose levels are not fully understood. Here, we examined the physiological role of Down syndrome critical region 1-4 (DSCR1-4), an endogenous calcineurin signaling inhibitor in the liver that mediates metabolic adaptation to fasting. METHODS: We assessed the effect of cyclosporine A, an inhibitor of calcineurin signaling on gluconeogenic gene expression in primary hepatocytes. DSCR1-4 expression was examined in diet- and genetically-induced mouse models of obesity. We also investigated the metabolic phenotype of a single extra copy of DSCR1-4 in transgenic mice and how DSCR1-4 regulates glucose homeostasis in the liver. RESULTS: Treatment with cyclosporin A increased hepatic glucose production and gluconeogenic gene expression. The expression of DSCR1-4 was induced by refeeding and overexpressed in obese mouse livers. Moreover, transgenic mice with a single extra copy of DSCR1-4 exhibited pyruvate intolerance and impaired glucose homeostasis. Mechanistically, DSCR1-4 overexpression increased phosphorylation of the cAMP response element-binding protein, which led to elevated expression levels of gluconeogenic genes and, thus, enhanced hepatic glucose production during fasting. CONCLUSION: A single extra copy of DSCR1-4 results in dysregulated hepatic glucose homeostasis and pyruvate intolerance. Our findings suggest that nutrient-sensitive DSCR1-4 is a novel target for controlling hepatic gluconeogenesis in diabetes.

mTOR controls ChREBP transcriptional activity and pancreatic ß cell survival under diabetic stress.

Impaired nutrient sensing and dysregulated glucose homeostasis are common in diabetes. However, how nutrient-sensitive signaling components control glucose homeostasis and ß cell survival under diabetic stress is not well understood. Here, we show that mice lacking the core nutrient-sensitive signaling component mammalian target of rapamycin (mTOR) in ß cells exhibit reduced ß cell mass and smaller islets. mTOR deficiency leads to a severe reduction in ß cell survival and increased mitochondrial oxidative stress in chemical-induced diabetes. Mechanistically, we find that mTOR associates with the carbohydrate-response element-binding protein (ChREBP)-Max-like protein complex and inhibits its transcriptional activity, leading to decreased expression of thioredoxin-interacting protein (TXNIP), a potent inducer of ß cell death and oxidative stress. Consistent with this, the levels of TXNIP and ChREBP were highly elevated in human diabetic islets and mTOR-deficient mouse islets. Thus, our results suggest that a nutrient-sensitive mTOR-regulated transcriptional network could be a novel target to improve ß cell survival and glucose homeostasis in diabetes.

S6K1 controls pancreatic ß cell size independently of intrauterine growth restriction.

Type 2 diabetes mellitus (T2DM) is a worldwide heath problem that is characterized by insulin resistance and the eventual loss of ß cell function. As recent studies have shown that loss of ribosomal protein (RP) S6 kinase 1 (S6K1) increases systemic insulin sensitivity, S6K1 inhibitors are being pursued as potential agents for improving insulin resistance. Here we found that S6K1 deficiency in mice also leads to decreased ß cell growth, intrauterine growth restriction (IUGR), and impaired placental development. IUGR is a common complication of human pregnancy that limits the supply of oxygen and nutrients to the developing fetus, leading to diminished embryonic ß cell growth and the onset of T2DM later in life. However, restoration of placental development and the rescue of IUGR by tetraploid embryo complementation did not restore ß cell size or insulin levels in S6K1-/- embryos, suggesting that loss of S6K1 leads to an intrinsic ß cell lesion. Consistent with this hypothesis, reexpression of S6K1 in ß cells of S6K1-/- mice restored embryonic ß cell size, insulin levels, glucose tolerance, and RPS6 phosphorylation, without rescuing IUGR. Together, these data suggest that a nutrient-mediated reduction in intrinsic ß cell S6K1 signaling, rather than IUGR, during fetal development may underlie reduced ß cell growth and eventual development of T2DM later in life.

Prognostic significance and function of phosphorylated ribosomal protein S6 in esophageal squamous cell carcinoma.

Ribosomal protein S6 is a key regulator of 40S ribosome biogenesis, and its phosphorylation is closely related to cell growth capacity. However, as a downstream target of S6 kinases, the clinical significance and the roles of S6 and S6 phosphorylation in cell viability and motility of esophageal squamous cell carcinoma remain unclear. Here, we show that high level of phosphorylated-ribosomal protein S6 (p-S6) (immunohistochemistry score ≥5) and an increased ratio of p-S6/S6 (immunohistochemistry score ≥0.75) were significantly associated with shortened disease-free survival in patients with esophageal squamous cell carcinoma in univariate analysis (P=0.049 and P<0.001, respectively). After adjusting for age, tumor-nodes-metastasis stage, chemotherapy, and radiation therapy in multivariate analysis, both p-S6 (hazard ratio 2.21, P=0.005) and p-S6/S6 (hazard ratio 2.40, P<0.001) remained independent adverse prognostic factors. In addition, S6 and S6 kinase 1 knockdown resulted in attenuation of viability by suppressing cyclin D1 expression in esophageal cancer cells. Furthermore, depletion of S6 and S6 kinase 1 resulted in a reduction in esophageal cancer cell migration and invasion. This was paralleled by a reduction in focal adhesion and by suppression of extracellular signal-regulated kinase and c-jun N-terminal kinase phosphorylation, which control cell motility. Collectively, these findings suggest that p-S6 and the ratio of p-S6/S6 are closely relevant to tumor progression and have prognostic significance in esophageal squamous cell carcinoma.

Clinicopathologic significance and function of mammalian target of rapamycin activation in esophageal squamous cell carcinoma.

Mammalian target of rapamycin (mTOR) has emerged as a key regulator of cell metabolism, growth, and proliferation. Despite the increasing significance of mTOR signaling in cancer cell cycle and proliferation, the clinical significance of activated mTOR in esophageal squamous cell carcinoma and its role in esophageal cancer cell proliferation and invasion remain unclear. Here, we show that both high levels of phosphorylated-mTOR and an increased ratio of phosphorylated-mTOR/mTOR (ratio ≥0.2) were significantly associated with shortened disease-specific survival in 165 patients with esophageal squamous cell carcinoma in univariate analysis (P = .047 for phosphorylated-mTOR, P = .021 for phosphorylated-mTOR/mTOR); phosphorylated-mTOR and phosphorylated-mTOR/mTOR remained independent prognostic factors after adjusting for age, TNM stage, chemotherapy, and radiation therapy in multivariate analysis (hazard ratio, 1.67, P = .025 for phosphorylated-mTOR; hazard ratio, 1.95, P = .006 for phosphorylated-mTOR/mTOR). Moreover, down-regulation of mTOR or mTOR complex components led to attenuation of proliferation, migration, and invasion of esophageal squamous cell carcinoma cell lines through suppression of cyclin D1 expression. Collectively, our findings suggest that phosphorylated-mTOR and the ratio of phosphorylated-mTOR/mTOR are closely linked to tumor progression and represent independent prognostic factors in esophageal squamous cell carcinoma, thereby providing a potential therapeutic target for this malignancy.