CRIP1 suppresses BBOX1-mediated carnitine metabolism to promote stemness in hepatocellular carcinoma.

Carnitine metabolism is thought to be negatively correlated with the progression of hepatocellular carcinoma (HCC) and the specific molecular mechanism is yet to be fully elucidated. Here, we report that little characterized cysteine-rich protein 1 (CRIP1) is upregulated in HCC and associated with poor prognosis. Moreover, CRIP1 promoted HCC cancer stem-like properties by downregulating carnitine energy metabolism. Mechanistically, CRIP1 interacted with BBOX1 and the E3 ligase STUB1, promoting BBOX1 ubiquitination and proteasomal degradation, and leading to the downregulation of carnitine. BBOX1 ubiquitination at lysine 240 is required for CRIP1-mediated control of carnitine metabolism and cancer stem-like properties. Further, our data showed that acetylcarnitine downregulation in CRIP1-overexpressing cells decreased beta-catenin acetylation and promoted nuclear accumulation of beta-catenin, thus facilitating cancer stem-like properties. Clinically, patients with higher CRIP1 protein levels had lower BBOX1 levels but higher nuclear beta-catenin levels in HCC tissues. Together, our findings identify CRIP1 as novel upstream control factor for carnitine metabolism and cancer stem-like properties, suggesting targeting of the CRIP1/BBOX1/ß-catenin axis as a promising strategy for HCC treatment.

Long non-coding RNA BBOX1-antisense RNA 1 enhances cell proliferation and migration and suppresses apoptosis in oral squamous cell carcinoma via the miR-3940-3p/laminin subunit gamma 2 axis.

Long non-coding RNAs (lncRNAs) play an essential role in oral squamous cell carcinoma (OSCC). We aimed to demonstrate the effects of lncRNA gamma-butyrobetaine hydroxylase 1 (BBOX1)-antisense RNA 1 (AS1) in OSCC and its regulatory mechanisms. The levels of BBOX1-AS1, microRNA (miR)-3940-3p, and laminin subunit gamma 2 (LAMC2) in OSCC were determined using reverse transcription-quantitative polymerase chain reaction. The correlations among BBOX1-AS1, miR-3940-3p, and LAMC2 were validated using luciferase, pull-down, and RNA immunoprecipitation assays. Cell proliferation, migration, and apoptosis were examined. BBOX1-AS1 and LAMC2 were notably overexpressed in OSCC, while miR-3940-3p showed the opposite trend. BBOX-1-AS1 silencing reduced the cell proliferation and migration, while promoting apoptosis. Mechanistically, BBOX1-AS1 modulates LAMC2 expression by competitively binding to miR-3940-3p. miR-3940-3p inhibition alleviated the inhibitory effects of BBOX1-AS1 deficiency on OSCC development. LAMC2 knockdown reversed these changes. Our results revealed that BBOX1-AS1 promotes the malignant phenotype of OSCC cells via the upregulation of LAMC2 expression by targeting miR-3940-3p, indicating that BBOX1-AS1 may be a novel target for OSCC intervention.

KLF5-induced BBOX1-AS1 contributes to cell malignant phenotypes in non-small cell lung cancer via sponging miR-27a-5p to up-regulate MELK and activate FAK signaling pathway.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a major histological subtype of lung cancer with high mortality and morbidity. A substantial amount of evidence demonstrates long non-coding RNAs (lncRNA) as critical regulators in tumorigeneis and malignant progression of human cancers. The oncogenic role of BBOX1 anti-sense RNA 1 (BBOX1-AS1) has been reported in several tumors. As yet, the potential functions and mechanisms of BBOX1-AS1 in NSCLC are obscure. METHODS: The gene and protein expression was detected by qRT-PCR and western blot. Cell function was determined by CCK-8, colony forming, would healing and transwell assays. Bioinformatics tools, ChIP assays, dual luciferase reporters system and RNA pull-down experiments were used to examine the interaction between molecules. Subcutaneous tumor models in nude mice were established to investigate in vivo NSCLC cell behavior. RESULTS: BBOX1-AS1 was highly expressed in NSCLC tissues and cells. High BBOX1-AS1 expression was associated with worse clinical parameters and poor prognosis. BBOX1-AS1 up-regulation was induced by transcription factor KLF5. BBOX1-AS1 deficiency resulted in an inhibition of cell proliferation, migration, invasion and EMT in vitro. Also, knockdown of BBOX1-AS1 suppressed NSCLC xenograft tumor growth in mice in vivo. Mechanistically, BBOX1-AS1 acted act as a competetive "sponge" of miR-27a-5p to promote maternal embryonic leucine zipper kinase (MELK) expression and activate FAK signaling. miR-27a-5p was confirmed as a tumor suppressor in NSCLC. Moreover, BBOX1-AS1-induced increase of cell proliferation, migration, invasion and EMT was greatly reversed due to the overexpression of miR-27a-5p. In addition, the suppressive effect of NSCLC progression owing to BBOX1-AS1 depletion was abated by the up-regulation of MELK. Consistently, BBOX1-AS1-mediated carcinogenicity was attenuated in NSCLC after treatment with a specific MELK inhibitor OTSSP167. CONCLUSIONS: KLF5-induced BBOX1-AS1 exerts tumor-promotive roles in NSCLC via sponging miR-27a-5p to activate MELK/FAK signaling, providing the possibility of employing BBOX1-AS1 as a therapeutic target for NSCLC patients.

Identification of BBOX1 as a Therapeutic Target in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease. Because of its heterogeneity and lack of hormone receptors or HER2 expression, targeted therapy is limited. Here, by performing a functional siRNA screening for 2-OG-dependent enzymes, we identified gamma-butyrobetaine hydroxylase 1 (BBOX1) as an essential gene for TNBC tumorigenesis. BBOX1 depletion inhibits TNBC cell growth while not affecting normal breast cells. Mechanistically, BBOX1 binds with the calcium channel inositol-1,4,5-trisphosphate receptor type 3 (IP3R3) in an enzymatic-dependent manner and prevents its ubiquitination and proteasomal degradation. BBOX1 depletion suppresses IP3R3-mediated endoplasmic reticulum calcium release, therefore impairing calcium-dependent energy-generating processes including mitochondrial respiration and mTORC1-mediated glycolysis, which leads to apoptosis and impaired cell-cycle progression in TNBC cells. Therapeutically, genetic depletion or pharmacologic inhibition of BBOX1 inhibits TNBC tumor growth in vitro and in vivo. Our study highlights the importance of targeting the previously uncharacterized BBOX1-IP3R3-calcium oncogenic signaling axis in TNBC. SIGNIFICANCE: We provide evidence from unbiased screens that BBOX1 is a potential therapeutic target in TNBC and that genetic knockdown or pharmacologic inhibition of BBOX1 leads to decreased TNBC cell fitness. This study lays the foundation for developing effective BBOX1 inhibitors for treatment of this lethal disease.This article is highlighted in the In This Issue feature, p. 1611.

19F NMR studies on γ-butyrobetaine hydroxylase provide mechanistic insights and suggest a dual inhibition mode.

The final step in the biosynthesis of l-carnitine in humans is catalysed by the 2-oxoglutarate and ferrous iron dependent oxygenase, γ-butyrobetaine hydroxylase (BBOX). 1H and 19F NMR studies inform on the BBOX mechanism including by providing evidence for cooperativity between monomers in substrate/some inhibitor binding. The value of the 19F NMR methods is demonstrated by their use in the design of new BBOX inhibitors.

Cation-π Interactions Contribute to Substrate Recognition in γ-Butyrobetaine Hydroxylase Catalysis.

γ-Butyrobetaine hydroxylase (BBOX) is a non-heme Fe(II) - and 2-oxoglutarate-dependent oxygenase that catalyzes the stereoselective hydroxylation of an unactivated C-H bond of γ-butyrobetaine (γBB) in the final step of carnitine biosynthesis. BBOX contains an aromatic cage for the recognition of the positively charged trimethylammonium group of the γBB substrate. Enzyme binding and kinetic analyses on substrate analogues with P and As substituting for N in the trimethylammonium group show that the analogues are good BBOX substrates, which follow the efficiency trend N(+) >P(+) >As(+). The results reveal that an uncharged carbon analogue of γBB is not a BBOX substrate, thus highlighting the importance of the energetically favorable cation-π interactions in productive substrate recognition.

Expression of genes involved in hepatic carnitine synthesis and uptake in dairy cows in the transition period and at different stages of lactation.

BACKGROUND: In rodents and pigs, it has shown that carnitine synthesis and uptake of carnitine into cells are regulated by peroxisome proliferator-activated receptor α (PPARA), a transcription factor which is physiologically activated during fasting or energy deprivation. Dairy cows are typically in a negative energy balance during early lactation. We investigated the hypothesis that genes of carnitine synthesis and uptake in dairy cows are enhanced during early lactation. RESULTS: mRNA abundances of PPARA and some of its classical target genes and genes involved in carnitine biosynthesis [trimethyllysine dioxygenase (TMLHE), 4-N-trimethylaminobutyraldehyde dehydrogenase (ALDH9A1), γ-butyrobetaine dioxygenase (BBOX1)] and uptake of carnitine [novel organic cation transporter 2 (SLC22A5)] as well as carnitine concentrations in liver biopsy samples of 20 dairy cows in late pregnancy (3 wk prepartum) and early lactation (1 wk, 5 wk, 14 wk postpartum) were determined. From 3 wk prepartum to 1 wk postpartum, mRNA abundances of PPARΑ and several PPARΑ target genes involved in fatty acid uptake, fatty acid oxidation and ketogenesis in the liver were strongly increased. Simultaneously, mRNA abundances of enzymes of carnitine synthesis (TMLHE: 10-fold; ALDH9A1: 6-fold; BBOX1: 1.8-fold) and carnitine uptake (SLC22A5: 13-fold) and the concentration of carnitine in the liver were increased from 3 wk prepartum to 1 wk postpartum (P < 0.05). From 1 wk to 5 and 14 wk postpartum, mRNA abundances of these genes and hepatic carnitine concentrations were declining (P < 0.05). There were moreover positive correlations between plasma concentrations of non-esterified fatty acids (NEFA) and hepatic carnitine concentrations at 1 wk, 5 wk and 14 wk postpartum (P < 0.05). CONCLUSIONS: The results of this study show for the first time that the expression of hepatic genes of carnitine synthesis and cellular uptake of carnitine is enhanced in dairy cows during early lactation. These changes might provide an explanation for increased hepatic carnitine concentrations observed in 1 wk postpartum and might be regarded as a physiologic means to provide liver cells with sufficient carnitine required for transport of excessive amounts of NEFA during a negative energy balance.

The effects of exercise training on γ-butyrobetaine hydroxylase and novel organic cation transporter-2 gene expression in the rat.

The concentration of carnitine in plasma is generally increased with exercise training, suggesting that either carnitine biosynthesis is stimulated or renal reabsorption of carnitine is enhanced, or both. Carnitine, an essential cofactor in the oxidation of fatty acids, is released into the plasma following hydroxylation by γ-butyrobetaine hydroxylase (BBH), the final enzyme in the biosynthetic pathway found primarily in the liver. The organic cation transporter (OCTN2), the carnitine transporter found in kidney, is important in the distribution of carnitine by facilitating its renal reabsorption from urine. In this study, we tested the hypothesis that exercise training increases gene and protein expression of BBH and OCTN2, resulting in enhanced plasma carnitine levels. Male Wistar rats were subjected to 2 daily exercise sessions of treadmill running, 5 days per week, for a 10-week period. The concentration of total carnitine in plasma was significantly increased in trained rats compared with sedentary rats. In trained rats, mRNA and protein expression of BBH were increased in liver, whereas only BBH mRNA expression was increased in kidney. Liver of trained rats demonstrated increased mRNA and protein expression of OCTN2 compared with sedentary rats. In kidney of trained rats, however, only an increase in mRNA expression of OCTN2 was observed. Our results suggest that the improved plasma carnitine status in the trained rat is associated with increased carnitine biosynthesis in liver and kidney. The observation that OCTN2 expression was increased in kidney suggests a potential role of the kidney in the reabsorption of carnitine from the urine.

The disruption of L-carnitine metabolism by aluminum toxicity and oxidative stress promotes dyslipidemia in human astrocytic and hepatic cells.

L-Carnitine is a critical metabolite indispensable for the metabolism of lipids as it facilitates fatty acid transport into the mitochondrion where ß-oxidation occurs. Human astrocytes (CCF-STTG1 cells) and hepatocytes (HepG2 cells) exposed to aluminum (Al) and hydrogen peroxide (H2O2), were characterized with lower levels of L-carnitine, diminished ß-oxidation, and increased lipid accumulation compared to the controls. γ-Butyrobetainealdehyde dehydrogenase (BADH) and butyrobetaine dioxygenase (BBDOX), two key enzymes mediating the biogenesis of L-carnitine, were sharply reduced during Al and H2O2 challenge. Exposure of the Al and H2O2-treated cells to α-ketoglutarate (KG), led to the recovery of L-carnitine production with the concomitant reduction in ROS levels. It appears that the channeling of KG to combat oxidative stress results in decreased L-carnitine synthesis, an event that contributes to the dyslipidemia observed during Al and H2O2 insults in these mammalian cells. Hence, KG may help alleviate pathological conditions induced by oxidative stress.

Activities of gamma-butyrobetaine dioxygenase and concentrations of carnitine in tissues of pigs.

In contrast to other species, less is known about carnitine homeostasis in the pig. This study was performed to yield information about the site of carnitine synthesis and carnitine concentrations in various tissues of pigs (Sus scrofa). We found that among several pig tissues, a considerable activity of gamma-butyrobetaine dioxygenase (BBD), the last enzyme of carnitine synthesis, exists, like in humans and several other species, only in liver and kidney. Activity of that enzyme in liver and kidney was lower at birth than in the subsequent weeks of life. Highest carnitine concentrations were found in skeletal muscle and heart. Carnitine concentrations in plasma, liver and kidney at birth were higher than in the subsequent weeks of life in spite of the low BBD activity at birth. In conclusion, this study shows that liver and kidney are the major sites of carnitine synthesis and that neonatal pigs do not have an insufficient carnitine status.

A moderate excess of dietary lysine lowers plasma and tissue carnitine concentrations in pigs.

This study was performed to investigate whether dietary lysine concentration influences the carnitine status of pigs. Therefore, an experiment with twenty young pigs with an average body weight of 21 kg was performed which were fed either a control diet (9.7 g lysine/kg) or a diet with a moderate excess of lysine (16.8 g lysine/kg). Concentrations of all the other amino acids did not differ between the diets. Pigs fed the high-lysine diet had lower concentrations of free and total carnitine in plasma, liver, kidney and skeletal muscle than control pigs (P<0.05). Pigs fed the high-lysine diet moreover had an increased concentration of trimethyllysine (TML), a reduced mRNA abundance of TML dioxygenase and reduced concentrations of gamma-butyrobetaine (BB) in muscle, indicating that the conversion of TML into BB in muscle was impaired. Concentrations of BB, the metabolic precursor of carnitine, in plasma, liver and kidney were also reduced in pigs fed the high-lysine diet while the activity of BB dioxygenase in kidney was not different and that in liver was even increased compared to control pigs (P<0.05). In conclusion, this study shows that a moderate dietary excess of lysine lowers plasma and tissue carnitine concentrations in pigs. Reduced concentrations of BB in liver and kidney suggest that the depressed carnitine status was likely caused by a decreased rate of carnitine synthesis due to a diminished availability of carnitine precursor, probably mainly as a result of an impaired BB formation in muscle.

Clofibrate treatment up-regulates novel organic cation transporter (OCTN)-2 in tissues of pigs as a model of non-proliferating species.

Recent studies have shown that treatment of rodents with agonists of peroxisome proliferator-activated receptor (PPAR)-alpha causes an up-regulation of novel organic cation transporter (OCTN)-2, a carnitine transporter, and increases carnitine concentration in the liver. This study was performed to investigate whether such effects occur also in pigs which like humans have a lower expression of PPAR alpha and are less responsive to treatment with PPAR alpha agonists than rodents. An experiment with 18 pigs was performed which were fed a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had higher relative mRNA concentrations of OCTN2 in liver (3.1-fold), skeletal muscle (1.5-fold) and epithelial cells from small intestine (1.8-fold) than control pigs (P<0.05). Pigs treated with clofibrate had also higher concentrations of free and total carnitine in the liver and a higher concentration of free carnitine in skeletal muscle than control pigs (P<0.05). Concentrations of gamma-butyrobetaine, the precursor of endogenous formation of carnitine, in liver, muscle and plasma did not differ between both groups; the activity of gamma-butyrobetaine dioxygenase, the rate limiting enzyme of carnitine synthesis, in the liver was lower in pigs treated with clofibrate than in control pigs (P<0.05). This study shows for the first time that treatment with a PPAR alpha agonist causes an up-regulation of OCTN2 in liver, muscle and enterocytes from small intestine of pigs. This in turn increases carnitine concentrations in liver and muscle probably by enhancing carnitine uptake into cells.

Treatment with pharmacological peroxisome proliferator-activated receptor alpha agonist clofibrate causes upregulation of organic cation transporter 2 in liver and small intestine of rats.

Activation of PPARalpha by clofibrate has recently been shown to cause upregulation of carnitine transporter organic cation transporter (OCTN) 2 and elevated carnitine concentrations in rat liver. The present study has been conducted to further explore the effect of clofibrate on OCTN expression, carnitine biosynthesis, and carnitine accumulation in different rat tissues, and thus two groups of rats were fed diets containing 0.5% clofibrate or 0% clofibrate (control group). PPARalpha-responsive genes were markedly upregulated in the liver (P<0.05), moderately in small intestine, but only slightly in other extrahepatic tissues by clofibrate. Relative mRNA concentration of OCTN2 in liver and small intestine was increased in rats fed clofibrate (P<0.05), whereas in other extrahepatic tissues mRNA concentration of OCTN2 did not differ between treatment groups. Concentration of total carnitine was higher in liver and small intestine but lower in plasma, kidney, and brain of rats fed clofibrate (P<0.05). Moreover, concentration of the carnitine precursor trimethyllysine and mRNA concentrations of specific genes involved in carnitine biosynthesis were increased in livers of rats fed clofibrate (P<0.05). The present study shows that clofibrate causes not only upregulation of OCTN2 in the liver but also in small intestine, and thus suggests that an increased intestinal absorption of carnitine might also contribute to the clofibrate-induced increase in hepatic carnitine concentration. Furthermore, the present results also indicate that an increased carnitine biosynthesis also contributes to the clofibrate-induced increase in hepatic carnitine concentration.

Mildronate, an inhibitor of carnitine biosynthesis, induces an increase in gamma-butyrobetaine contents and cardioprotection in isolated rat heart infarction.

The inhibition of gamma-butyrobetaine (GBB) hydroxylase, a key enzyme in the biosynthesis of carnitine, contributes to lay ground for the cardioprotective mechanism of action of mildronate. By inhibiting the biosynthesis of carnitine, mildronate is supposed to induce the accumulation of GBB, a substrate of GBB hydroxylase. This study describes the changes in content of carnitine and GBB in rat plasma and heart tissues during long-term (28 days) treatment of mildronate [i.p. (intraperitoneal) 100 mg/kg/daily]. Obtained data show that in concert with a decrease in carnitine concentration, the administration of mildronate caused a significant increase in GBB concentration. We detected about a 5-fold increase in GBB contents in the plasma and brain and a 7-fold increase in the heart. In addition, we tested the cardioprotective effect of mildronate in isolated rat heart infarction model after 3, 7, and 14 days of administration. We found a statistically significant decrease in necrotic area of infarcted rat hearts after 14 days of treatment with mildronate. The cardioprotective effect of mildronate correlated with an increase in GBB contents. In conclusion, our study, for the first time, provides experimental evidence that the long-term administration of mildronate not only decreases free carnitine concentration, but also causes a significant increase in GBB concentration, which correlates with the cardioprotection of mildronate.