Sex-specific survival gene mutations are discovered as clinical predictors of clear cell renal cell carcinoma.

Although sex differences have been reported in patients with clear cell renal cell carcinoma (ccRCC), biological sex has not received clinical attention and genetic differences between sexes are poorly understood. This study aims to identify sex-specific gene mutations and explore their clinical significance in ccRCC. We used data from The Cancer Genome Atlas-Kidney Renal Clear Cell Carcinoma (TCGA-KIRC), The Renal Cell Cancer-European Union (RECA-EU) and Korean-KIRC. A total of 68 sex-related genes were selected from TCGA-KIRC through machine learning, and 23 sex-specific genes were identified through verification using the three databases. Survival differences according to sex were identified in nine genes (ACSS3, ALG13, ASXL3, BAP1, JADE3, KDM5C, KDM6A, NCOR1P1, and ZNF449). Female-specific survival differences were found in BAP1 in overall survival (OS) (TCGA-KIRC, p = 0.004; RECA-EU, p = 0.002; and Korean-KIRC, p = 0.003) and disease-free survival (DFS) (TCGA-KIRC, p = 0.001 and Korean-KIRC, p = 0.000004), and NCOR1P1 in DFS (TCGA-KIRC, p = 0.046 and RECA-EU, p = 0.00003). Male-specific survival differences were found in ASXL3 (OS, p = 0.017 in TCGA-KIRC; and OS, p = 0.005 in RECA-EU) and KDM5C (OS, p = 0.009 in RECA-EU; and DFS, p = 0.016 in Korean-KIRC). These results suggest that biological sex may be an important predictor and sex-specific tailored treatment may improve patient care in ccRCC.

Discovery and Validation of Survival-Specific Genes in Papillary Renal Cell Carcinoma Using a Customized Next-Generation Sequencing Gene Panel.

PURPOSE: Papillary renal cell carcinoma (PRCC), the second most common kidney cancer, is morphologically, genetically, and molecularly heterogeneous with diverse clinical manifestations. Genetic variations of PRCC and their association with survival are not yet well-understood. This study aimed to identify and validate survival-specific genes in PRCC and explore their clinical utility. MATERIALS AND METHODS: Using machine learning, 293 patients from the Cancer Genome Atlas-Kidney Renal Papillary Cell Carcinoma (TCGA-KIRP) database were analyzed to derive genes associated with survival. To validate these genes, DNAs were extracted from the tissues of 60 Korean PRCC patients. Next generation sequencing was conducted using a customized PRCC gene panel of 202 genes, including 171 survival-specific genes. Kaplan-Meier and Log-rank tests were used for survival analysis. Fisher's exact test was performed to assess the clinical utility of variant genes. RESULTS: A total of 40 survival-specific genes were identified in the TCGA-KIRP database through machine learning and statistical analysis. Of them, 10 (BAP1, BRAF, CFDP1, EGFR, ITM2B, JAK1, NODAL, PCSK2, SPATA13, and SYT5) were validated in the Korean-KIRP database. Among these survival gene signatures, three genes (BAP1, PCSK2, and SPATA13) showed survival specificity in both overall survival (OS) (p = 0.00004, p = 1.38 × 10-7, and p = 0.026, respectively) and disease-free survival (DFS) (p = 0.00002, p = 1.21 × 10-7, and p = 0.036, respectively). Notably, the PCSK2 mutation demonstrated survival specificity uniquely in both the TCGA-KIRP (OS: p = 0.010 and DFS: p = 0.301) and Korean-KIRP (OS: p = 1.38 × 10-7 and DFS: p = 1.21 × 10-7) databases. CONCLUSIONS: We discovered and verified genes specific for the survival of PRCC patients in the TCGA-KIRP and Korean-KIRP databases. The survival gene signature, including PCSK2 commonly obtained from the 40 gene signature of TCGA and the 10 gene signature of the Korean database, is expected to provide insight into predicting the survival of PRCC patients and developing new treatment.

Identification of Survival-Specific Genes in Clear Cell Renal Cell Carcinoma Using a Customized Next-Generation Sequencing Gene Panel.

PURPOSE: Although mutations are associated with carcinogenesis, little is known about survival-specific genes in clear cell renal cell carcinoma (ccRCC). We developed a customized next-generation sequencing (NGS) gene panel with 156 genes. The purpose of this study was to investigate whether the survival-specific genes we found were present in Korean ccRCC patients, and their association with clinicopathological findings. MATERIALS AND METHODS: DNA was extracted from the formalin-fixed, paraffin-embedded tissue of 22 ccRCC patients. NGS was performed using our survival-specific gene panel with an Illumina MiSeq. We analyzed NGS data and the correlations between mutations and clinicopathological findings and also compared them with data from the Cancer Genome Atlas-Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) and Renal Cell Cancer-European Union (RECA-EU). RESULTS: We found a total of 100 mutations in 37 of the 156 genes (23.7%) in 22 ccRCC patients. Of the 37 mutated genes, 11 were identified as clinicopathologically significant. Six were novel survival-specific genes (ADAMTS10, CARD6, NLRP2, OBSCN, SECISBP2L, and USP40), and five were top-ranked mutated genes (AKAP9, ARID1A, BAP1, KDM5C, and SETD2). Only CARD6 was validated as an overall survival-specific gene in this Korean study (p = 0.04, r = -0.441), TCGA-KIRC cohort (p = 0.0003), RECA-EU (p = 0.0005). The 10 remaining gene mutations were associated with clinicopathological findings; disease-free survival, mortality, nuclear grade, sarcomatoid component, N-stage, sex, and tumor size. CONCLUSIONS: We discovered 11 survival-specific genes in ccRCC using data from TCGA-KIRC, RECA-EU, and Korean patients. We are the first to find a correlation between CARD6 and overall survival in ccRCC. The 11 genes, including CARD6, NLRP2, OBSCN, and USP40, could be useful diagnostic, prognostic, and therapeutic markers in ccRCC.

Effects of Toona sinensis leaf extract on lipolysis in differentiated 3T3-L1 adipocytes.

The effect of substances extracted from Toona sinensis leaves with 50% alcohol solution on lipolysis was investigated in cultured 3T3-L1 differentiated adipocytes. The amount of glycerol released from cells into culture medium was used to measure lipolysis activity. Glycerol release was increased by Toona sinensis leaf extract in a dose-dependent and time-dependent manner. Following treatment of 3T3-L1 adipocyte cells with various concentrations of Toona sinensis leaf extract (0.001, 0.01, and 0.1 mg/mL) for 6 hours, the amounts of glycerol released from 3T3-L1 cells increased from a control value of 99 nmol/mg protein to 127, 144, and 154 nmol/mg protein, respectively. The lipolytic effect of Toona sinensis leaf extract was not inhibited by pretreatment of cells with cycloheximide, econazole, baicalein, or indomethacin. However, the lipolytic activity induced by Toona sinensis leaf extract was diminished by dibutyryl cyclic adenosine-5'-monophosphate (dibutyryl cAMP) and the protein kinase C inhibitor calphostin C. These results indicate that the lipolytic effect induced by Toona sinensis leaf substances may be involved in the protein kinase C pathway and may be down-regulated by cAMP.

Enhancement of glucose uptake in 3T3-L1 adipocytes by Toona sinensis leaf extract.

The effects of substances extracted from Toona sinensis leaves, using 50% alcohol/water, on cellular [3H]-2-deoxyglucose uptake in differentiated cultured 3T3-L1 adipocytes were investigated. Following treatment of cells with 0.001, 0.01, or 0.1 mg/mL extracts for 60 minutes, [3H]-2-deoxyglucose uptake increased from a basal value of 0.23 nmol/min/mg protein to 0.30, 0.33, and 0.38 nmol/min/mg protein, respectively. In insulin-stimulated cells, cellular [3H]-2-deoxyglucose uptake was enhanced by Toona sinensis leaf extract from a basal value of 0.35 nmol/min/mg protein to 0.41, 0.46, and 0.52 nmol/min/mg protein, respectively. Cellular glucose uptake was also enhanced by Toona sinensis leaf extract after incubation of cells with 20 mM glucose for 48 hours. Cellular glucose uptake with a combination of Toona sinensis leaf extract and insulin was significantly inhibited by pretreatment of cells with the protein synthesis inhibitor cycloheximide and the protein kinase C inhibitor calphostin C in normal-, medium- and high-glucose media. However, the glucose uptake-enhancing effect of Toona sinensis leaf extract was not diminished by cycloheximide and calphostin C in the absence of insulin. These results indicate that enhancement of cellular glucose uptake by Toona sinensis leaf extract in basal and insulin-stimulated 3T3-L1 adipocytes may be mediated by distinct mechanisms.

Effects of crude drugs on lipolysis in differentiated 3T3-L1 adipocytes.

In the present study, aqueous fractions extracted from Radix Ginseng, Radix Rehmanniae, Radix Puerariae, Radix Asparagi, Cortex Phellodendri and Radix Scutellariae were investigated for their effects on lipolysis measured the glycerol release in cultured 3T3-L1 differentiated adipocytes cells. Following treatment of cells with various concentrations of water-soluble extracts ranging from 0.1, 1 to 10 mg/ml for 60 mim, the basal glycerol release from 3T3-L1 cells was changed from 71 nmole/mg protein of control to 48, 46 and 31 nmole/mg protein in Radix Ginseng-treated cells. Amount of glycerol was reduced to 60, 26 and 20 by Radix Rehmanniae. In the presence of Radix Puerariae, glycerol release was decreased to 35, 34 and 30, respectively. After exposure to Radix Asparagi, amount of glycerol became 108, 73 and 70 nmole/mg protein, respectively. In the case of Cortex Phellodendri, amount of glycerol was increased from 126, 112 to 90, respectively. In the presence of Radix Scutellariae, the glycerol was changed to 118, 77 and 29, respectively. In isoproterenol-stimulated cells, the glycerol release from cells by Radix Ginseng was changed from 169 of control to 76, 73 and 72 nmole/mg protein, respectively. After incubation with Radix Rehmanniae, amount of glycerol decreased to 52, 35 and 11, respectively. In the presence of Radix Puerariae, the glycerol was changed to 26, 25 to 20, respectively. In the presence of Radix Asparagi, the glycerol became 160, 96 and 64, respectively. In the case of Cortex Phellodendri, the glycerol was increased to 160, 92 to 88, respectively. In the presence of Radix Scutellariae, the glycerol was changed to 149, 83 and 50, respectively. These results indicated that the water-soluble substances from Radix Ginseng, Radix Rehmanniae and Radix Puerariae decreased the lipolysis in basal and isoproterenol-stimulated 3T3-L1 adipocytes.

Possible mechanism of endothelin-induced Ca2+ mobility in A7r5 cultured vascular smooth muscle cells.

We studied the mechanism by which endothelin-1 (ET-1) affects the mobility of intracellular free Ca2+ ([Ca2+]i) in cultured A7r5 aortic smooth muscle cells. ET-1 at 10(-9) to 10(-7) M increased [Ca2+]i in Ca2+-containing buffer and Ca2+-free buffer. Pretreatment with ET-1 inhibited thapsigargin- and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced [Ca2+]i increases in Ca2+-free buffer. Pretreatment with thapsigargin and CCCP partially abolished the [Ca2+]i increase induced by ET-1. The ET-1-induced Ca2+ signal was partially suppressed by the ETA receptor antagonist BQ123 and the ETB receptor antagonist BQ788 and nifedipine. Pretreatment of cells with the phospholipase C inhibitor U73122 reduced the ET-1-induced [Ca2+]i increase. These results suggest that the ET-1-induced [Ca2+]i increase in A7r5 smooth muscle cells initially activates the ETA receptor, leading to Ca2+ influx and increased internal Ca2+ release from endoplasmic reticulum and mitochondrial Ca2+ stores. The ETB receptor and L-type Ca2+ channel are involved in maintaining further extracellular Ca2+ influx. ET-1-induced intracellular Ca2+ release was also modulated by phospholipase C-coupled events.