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Background The Enoyl-CoA hydratase/isomerase family plays a crucial role in the metabolism of tumors, being crucial for maintaining the energy balance and biosynthetic needs of cancer cells. However, the enzymes within this family that are pivotal in gastric cancer (GC) remain unclear. Methods We employed bioinformatics techniques to identify key Enoyl-CoA hydratase/isomerase in GC. The expression of ECHDC2 and its clinical significance were validated through tissue microarray analysis. The role of ECHDC2 in GC was further assessed using colony formation assays, CCK8 assay, EDU assay, Glucose and lactic acid assay, and subcutaneous tumor experiments in nude mice. The mechanism of action of ECHDC2 was validated through Western blotting, Co-immunoprecipitation, and immunofluorescence experiments. Results Our analysis of multiple datasets indicates that low expression of ECHDC2 in GC is significantly associated with poor prognosis. Overexpression of ECHDC2 notably inhibits aerobic glycolysis and proliferation of GC cells both in vivo and in vitro. Further experiments revealed that overexpression of ECHDC2 suppresses the P38 MAPK pathway by inhibiting the protein level of MCCC2, thereby restraining glycolysis and proliferation in GC cells. Ultimately, it was discovered that ECHDC2 promotes the ubiquitination and subsequent degradation of MCCC2 protein by binding with NEDD4. Conclusions These findings underscore the pivotal role of the ECHDC2 in regulating aerobic glycolysis and proliferation in GC cells, suggesting ECHDC2 as a potential therapeutic target in GC.

Prostate cancer (PCa) is the most common cancer in American men, and the mechanisms of development and progression are still not completely clear. Methylcrotonoyl-CoA carboxylase 2 (MCCC2) was previously identified overexpressed in PCa with lymph node metastasis, but its specific role and mechanisms need further investigation. This study aimed to investigate the role of MCCC2 in PCa cells and its underlying mechanisms. Quantitative RT-PCR and Western blotting were used to detect MCCC2 mRNA and protein expression in normal prostate epithelium and cancerous cells. Upon manipulation of MCCC2 expression, cell proliferation was measured by CCK-8 assays and migration and invasion were determined by transwell assays. Changes of apoptosis, cell cycle and mitochondrial membrane potential were evaluated by flow cytometry. MCCC2-mediated signaling pathways were screened by bioinformatics and verified by RT-PCR and Western blotting. Finally, immunohistochemistry was performed to detect the expression of MCCC2 and glutamate dehydrogenase 1 (GLUD1) in PCa tissues to analyze their correlation. We demonstrated that MCCC2 promoted cell proliferation, migration and invasion but inhibited apoptosis in PCa cells. In addition, MCCC2 in 22Rv1 cells induced mitochondrial damage. In PCa tissues, MCCC2 overexpression associated with lymph node metastasis ( =0.001) and high Gleason scores ( <0.001). MCCC2 positively correlated with GLUD1 expression in PCa tissues (r=0.435, <0.001). Ectopic overexpression of MCCC2 up-regulated GLUD1 and p38 MAPK expression, whereas inhibition of MCCC2 decreased GLUD1 and p38 MAPK expression. MCCC2 exerts oncogenic function in PCa through regulating GLUD1-p38 MAPK signaling pathway, and it may be a potential treatment target.

Background The mitochondrial gene MCCC2, a subunit of the heterodimer of 3-methylcrotonyl-CoA carboxylase, plays a pivotal role in catabolism of leucine and isovaleric acid. The molecular mechanisms and prognostic value still need to be explored in the context of specific cancers, including colorectal cancer (CRC). Methods In vitro and in vivo cell-based assays were performed to explore the role of MCCC2 in CRC cell proliferation, invasion, and migration. Mitochondrial morphology, membrane potential, intracellular reactive oxygen species (ROS), telomerase activity, and telomere length were examined and analyzed accordingly. Protein complex formation was detected by co-immunoprecipitation (CO-IP). Mitochondrial morphology was observed by transmission electron microscopy (TEM). The Cancer Genome Atlas (TCGA) CRC cohort analysis, qRT-PCR, and immunohistochemistry (IHC) were used to examine the MCCC2 expression level. The association between MCCC2 expression and various clinical characteristics was analyzed by chi-square tests. CRC patients’ overall survival (OS) was analyzed by Kaplan–Meier analysis. Results Ectopic overexpression of MCCC2 promoted cell proliferation, invasion, and migration, while MCCC2 knockdown (KD) or knockout (KO) inhibited cell proliferation, invasion, and migration. MCCC2 KD or KO resulted in reduced mitochondria numbers, but did not affect the gross ATP production in the cells. Mitochondrial fusion markers MFN1, MFN2, and OPA1 were all upregulated in MCCC2 KD or KO cells, which is in line with a phenomenon of more prominent mitochondrial fusion. Interestingly, telomere lengths of MCCC2 KD or KO cells were reduced more than control cells. Furthermore, we found that MCCC2 could specifically form a complex with telomere binding protein TRF2, and MCCC2 KD or KO did not affect the expression or activity of telomerase reverse transcriptase (TERT). Finally, MCCC2 expression was heightened in CRC, and patients with higher MCCC2 expression had favorable prognosis. Conclusions Together, we identified MCCC2 as a novel mediator between mitochondria and telomeres, and provided an additional biomarker for CRC stratification.

SIRT4 is well-known as a tumor suppressor by controlling several metabolic pathways, although it is highly expressed in certain cancers including hepatocellular carcinoma (HCC). Here, we reported that SIRT4 was highly expressed in the voltage-gated calcium channel α2δ1 subunit-positive HCC tumor-initiating cells (TIC), and was upregulated by α2δ1-mediated calcium signaling. Moreover, the expression of SIRT4 in HCC tissues was predictive of poor prognosis of the patients. Interestingly, SIRT4 was functionally sufficient and indispensable to promote TIC properties and invasiveness of HCC cells by directly deacetylating the leucine catabolism pathway enzyme-3-methylcrotonyl-CoA carboxylase 2 (MCCC2) at K269, leading to the formation of a stable MCCC1/MCCC2 complex with robust MCCC enzymatic activity to produce more acetyl-CoA, which resulted in increased H3K27 acetylation and stem cell-like properties at doses≤2 µM. However, 10 µM acetyl-CoA was neither able to enhance H3K27 acetylation, nor to promote stem cell-like properties, while forced expression of SIRT4 in α2δ1 cells resulted in retardation of tumor growth . Thus, SIRT4 serves as an oncogene to promote stemness and invasiveness by controlling the production of acetyl-CoA, linking α2δ1-mediated calcium signaling to SIRT4-mediated epigenetic reprogramming of HCC TICs which hold significant potential for the development of novel therapeutic strategies targeting TICs, and the dual roles of SIRT4 in HCC might be dependent on the production levels of acetyl-CoA.

Background The role of methylcrotonoyl-CoA carboxylase 2 (MCCC2) in the development of tumors is well-established, and the involvement of leucine in the liver is well-known. However, the role of MCCC2 and the correlation between MCCC2 and leucine in the progression of hepatocellular carcinoma (HCC) have not yet been reported. Methods In this study, the Gepia database was used to evaluate the prognostic value of MCCC2 in HCC. The expression and localization of MCCC2 in HCC cells were determined by western blot and immunofluorescence assays. Flow cytometry and CCK-8 and transwell assays were carried out to explore the effect of MCCC2 on cell proliferation, migration, and invasion. In addition, mass spectrometry analysis was used to predict the potential cell function of MCCC2 in HCC. Results We found that the expression of MCCC2 increased in HCC tissues and that high expression of MCCC2 could predict poor outcomes in HCC patients. Knockdown expression of MCCC2 in HCC cells could reduce cell proliferation, migration, and invasion ability in vitro and could inhibit HCC cell proliferation in vivo. Interestingly, we found that HCC cells transfected with MCCC2-sgRNA failed to respond to leucine deprivation. Meanwhile, leucine deprivation inhibited cell proliferation, migration, and invasion in HCC cells where MCCC2 was present rather than in cells where MCCC2 was absent. In addition, knockdown of MCCC2 significantly reduced the glycolysis markers, glucose consumption, lactate secretion, and acetyl-CoA level, which is a product of leucine metabolism. Furthermore, we found that MCCC2 promotes the activation of ERK. Profiling the MCCC2 binding proteins revealed that MCCC2-associated proteins are enriched in biological processes, such as protein metabolism, energy pathway, and metabolism in HCC cells. Conclusions Our findings revealed that MCCC2 plays a critical role in the development of HCC, and the leucine metabolism pathway might be a novel target in HCC treatment.

Background Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine metabolism caused by mutations in MCCC1 or MCCC2 encoding the α and β subunit of MCC, respectively. The phenotype is highly variable ranging from acute neonatal onset with fatal outcome to asymptomatic adults. Methods We report clinical, biochemical, enzymatic and mutation data of 88 MCC deficient individuals, 53 identified by newborn screening, 26 diagnosed due to clinical symptoms or positive family history and 9 mothers, identified following the positive newborn screening result of their baby. Results Fifty-seven percent of patients were asymptomatic while 43% showed clinical symptoms, many of which were probably not related to MCC deficiency but due to ascertainment bias. However, 12 patients (5 of 53 identified by newborn screening) presented with acute metabolic decompensations. We identified 15 novel MCCC1 and 16 novel MCCC2 mutant alleles. Additionally, we report expression studies on 3 MCCC1 and 8 MCCC2 mutations and show an overview of all 132 MCCC1 and MCCC2 variants known to date. Conclusions Our data confirm that MCC deficiency, despite low penetrance, may lead to a severe clinical phenotype resembling classical organic acidurias. However, neither the genotype nor the biochemical phenotype is helpful in predicting the clinical course.

Methylcrotonoyl-CoA carboxylase 2 (MCCC2), a subunit of 3-Methylcrotonyl-CoA carboxylase (MCC), is reported to be involved in tumor formation and development. However, the role of MCCC2 in breast cancer is unknown. MCCC2 expression was examined in 138 cases of breast cancer and matched adjacent normal tissues by quantitative reverse transcription PCR and immunohistochemistry. The influence of MCCC2 expression on cell proliferation was evaluated by CCK-8 and colony formation assay. Quantitative reverse transcription PCR results show MCCC2 mRNA levels were significantly greater in breast cancer tissues than normal tissues (p < 0.05). Immunohistochemistry analysis revealed that MCCC2 overexpression was significantly associated with Tumor, Node, Metastasis stage and lymph node metastasis and predicted an unfavorable prognosis (p < 0.05). CCK-8 and colony formation assay indicated that MCCC2 overexpression significantly promoted cell proliferation. These data indicate MCCC2 overexpression predicts an unfavorable prognosis and promotes cell proliferation in breast cancer, which may serve as a potential prognostic biomarker.

New technology enables expansion of newborn screening (NBS) of inborn errors aimed to prevent adverse outcome. In conditions with a large share of asymptomatic phenotypes, the potential harm created by NBS must carefully be weighed against benefit. Policies vary throughout the United States, Australia, and Europe due to limited data on outcome and treatability of candidate screening conditions. We elaborated the rationale for decision making in 3‐methylcrotonyl‐coenzyme A (CoA) carboxylase deficiency (MCCD), which afflicts leucine catabolism, with reported outcomes ranging from asymptomatic to death. In Bavaria, we screened 677,852 neonates for 25 conditions, including MCCD, based on elevated concentrations of 3‐hydroxyisovalerylcarnitine (3‐HIVA‐C). Genotypes of MCCA (MCCC1) and MCCB (MCCC2) were assessed in identified newborns, their relatives, and in individuals (n=17) from other regions, and correlated to biochemical and clinical phenotypes. NBS revealed eight newborns and six relatives with MCCD, suggesting a higher frequency than previously assumed (1:&!sol;84,700). We found a strikingly heterogeneous spectrum of 22 novel and eight reported mutations. Allelic variants were neither related to biochemical nor anamnestic data of our probands showing all asymptomatic or benign phenotypes. Comparative analysis of case reports with NBS data implied that only few individuals (<10%) develop symptoms. In addition, none of the symptoms reported so far can clearly be attributed to MCCD. MCCD is a genetic condition with low clinical expressivity and penetrance. It largely represents as nondisease. So far, there are no genetic or biochemical markers that would identify the few individuals potentially at risk for harmful clinical expression. The low ratio of benefit to harm was pivotal to the decision to exclude MCCD from NBS in Germany. MCCD may be regarded as exemplary of the ongoing controversy arising from the inclusion of potentially asymptomatic conditions, which generates a psychological burden for afflicted families and a financial burden for health care systems. Hum Mutat 27(8), 748–759, 2006. © 2006 Wiley‐Liss, Inc.

Methylcrotonylglycinuria (MCG) is an inborn error of leucine catabolism and results from the deficiency of 3-methylcrotonyl-CoA carboxylase. Patients with MCG show a highly variable clinical phenotype, ranging from asymptomatic to severe. With the introduction of newborn screening using tandem mass spectrometry, most patients with MCG are identified in their asymptomatic neonatal periods. Owing to their fair clinical outcomes, there exists a controversy over the need for aggressive medical intervention or even for newborn screening for MCG. Our study, reporting 11 Korean MCG patients from nine unrelated families, who were identified by newborn screening or family member testing and normally developed without experiencing an metabolic crisis during the follow-up period of 2.6±1.96 years (range, 1-10 years), indicates that the aggressive medical intervention might not be needed at least for the MCG patients identified by screening program in asymptomatic period. A total of six MCCC2 mutations, but no MCCC1 mutation, were identified in 17 of 18 alleles (94.4%). p.D280Y was identified in the 12/18 alleles (66.7%), indicating a founder effect. Moreover, the rest five variants, p.S342K, p.Q496H, p.P552S, p.T556A and p.P459S, were all previously unreported. The results of our study indicate that the distinct molecular genetic characteristics exist in Korean MCG patients.