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Background Previously, we performed analysis of gene expression in 46 axillary lymph node negative tumors and identified molecular gene signatures that resulted in different clinical outcomes. The aim of this study was to determine the correlation of γ-glutamyl hydrolase (GGH), fatty acid amide hydrolase (FAAH), Pirin (PIR) and TAF5-like RNA polymerase II, p300/CBP-associated factor (PCAF)-associated factor, 65 kDa (TAF5L), selected from identified gene signatures, with clinical outcomes as well as classical clinicopathological characteristics in primary invasive breast cancer patients. Methods The protein levels of GGH, FAAH, PIR and TAF5L were assessed by immunohistochemistry (IHC) on a panel of 80 primary invasive breast tumors. Quantitative real-time PCR (qRT-PCR) and western blot analysis were performed to verify the expression levels of the candidate biomarkers. Patient disease-specific survival (DSS) and recurrence-free survival (RFS) were evaluated using the Kaplan-Meier method. The prognostic biomarkers were identified by univariate analysis with a log-rank test and by multivariate analysis with Cox proportional hazards regression models. Results The GGH and FAAH protein levels were significantly up-regulated in invasive breast cancer tumors compared with adjacent non-cancerous tissues. Furthermore, the protein levels of GGH and FAAH were significantly correlated in tumor tissues. Tumoral GGH protein expression was significantly correlated with shorter DSS and RFS. Furthermore, the protein expression of GGH was positively correlated with undifferentiated tumors (BRE grade III) and ER/PR expressing tumors. Multivariate regression analysis showed that only GGH protein expression independently predicts DSS. No such correlations were found for FAAH, PIR and TAF5L protein expression. However, elevated protein levels of FAAH were positively associated with high number of lymph node involvement and upregulated levels of PIR were positively related with lymph node metastasis. The TAF5L was pronouncedly down-regulated in primary invasive breast cancer tissues compared to matched adjacent non-cancerous tissues. Conclusion These data show for the first time that cytoplasmic GGH might play a relevant role in the development and progression of invasive breast cancer, warranting further investigations. Our findings suggest that GGH serve as a potential biomarker of unfavorable clinical outcomes over short-term follow-up in breast cancer. The GGH may be a very attractive targeted therapy for selected patients.

Carboxypeptidase G2 (CPG2) is a bacterial enzyme widely used to detoxify methotrexate (MTX) and in enzyme/prodrug therapy for cancer treatment. However, several drawbacks, such as instability, have limited its efficiency. Herein, we have evaluated the properties of a putative CPG2 from Acinetobacter sp. 263903-1 (AcCPG2). AcCPG2 is compared with a CPG2 derived from Pseudomonas sp. strain RS-16 (PsCPG2), available as an FDA-approved medication called glucarpidase. After modeling AcCPG2 using the I-TASSER program, the refined model was validated by PROCHECK, VERIFY 3D and according to the Z score of the model. Using computational analyses, AcCPG2 displayed higher thermodynamic stability and a lower aggregation propensity than PsCPG2. AcCPG2 showed an optimum pH of 7.5 against MTX and was stable over a pH range of 5–10. AcCPG2 exhibited optimum activity at 50 °C and higher thermal stability at a temperature range of 20–70 °C compared to PsCPG2. The Km value of the purified AcCPG2 toward folate and MTX was 31.36 µM and 44.99 µM, respectively. The Vmax value of AcCPG2 for folate and MTX was 125.80 µmol/min/mg and 48.90 µmol/min/mg, respectively. Accordingly, thermostability and pH versatility makes AcCPG2 a potential biobetter variant for therapeutic applications.

Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to redesign any chosen enzyme to be similarly stimulus responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for proenzyme design. For a model system, we employ carboxypeptidase G2 (CPG2), a clinically approved enzyme that has applications in both the treatment of cancer and controlling drug toxicity. Detailed kinetic characterization of the most effectively designed variants shows that they are inhibited by ∼80% compared to the unmodified protein, and their activity is fully restored following incubation with site-specific proteases. Introducing disulfide bonds between the pro- and catalytic domains based on the design models increases the degree of inhibition to 98% but decreases the degree of restoration of activity by proteolysis. A selected disulfide-containing proenzyme exhibits significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization provides detailed insights into the prodomain binding and inhibition mechanisms. The described methodology is general and could enable the design of a variety of proproteins with precise spatial regulation.

PurposeThe enzymes gamma-glutamyl hydrolase (GGH) and folylpolyglutamate synthetase (FPGS) regulate intracellular folate concentrations needed for cell proliferation, DNA synthesis, and repair. High GGH expression affects 5-FU thymidylate synthase (TS) inhibition and is a risk factor for various malignancies. Here, the clinical significance of GGH and FPGS expression was investigated in Stage II/III gastric cancer patients undergoing postoperative adjuvant chemotherapy with S-1.MethodsSurgical specimens of cancer tissue and adjacent normal mucosa, obtained from 253 patients with previously untreated gastric cancer, were examined. GGH and FPGS mRNA expression was measured by qPCR to evaluate their clinicopathological significance in gastric cancer patients after curative resection.ResultsWhile FPGS expression showed no significant differences between the cancerous and normal samples, GGH expression was higher in cancer tissue than in adjacent normal mucosa. High GGH expression was correlated with age, histological type, and vascular invasion. Overall survival (OS) of patients with high GGH mRNA expression was significantly poorer than of patients with low GGH expression. Multivariate analysis showed that high GGH expression was an independent prognostic factor of OS (HR: 2.58, 95% CI 1.29–5.16). Patients who received S-1 adjuvant treatment showed a significantly poor OS between high GGH/low FPGS and low GGH/high FPGS. Patients without adjuvant treatment showed no significant difference.ConclusionGGH expression was significantly higher in gastric cancer tissue than in adjacent normal mucosa. High GGH and low FPGS expression is a useful independent predictor of poor outcomes in stage II/III gastric cancer patients undergoing postoperative adjuvant chemotherapy with S-1.

To establish the individualized treatment of patients with colorectal cancer, factors associated with chemotherapeutic effects should be identified. However, to the best of our knowledge, few studies are available on this topic, although it is known that the prognosis of patients and sensitivity to chemotherapy depend on the location of the tumor and that the tumor location is important for individualized treatment. In this study, primary tumors obtained from 1,129 patients with colorectal cancer were used to measure the mRNA expression levels of the following genes associated with the effects of standard chemotherapy for colorectal cancer: 5-fluorouracil (5-FU)-related thymidylate synthase (TYMS), dihydropyrimidine dehydrogenase (DPYD) and thymidine phosphorylase (TYMP); folate-related dihydrofolate reductase (DHFR), folylpolyglutamate synthase (FPGS) and gamma-glutamyl hydrolase (GGH); irinotecan-related topoisomerase I (TOP1); oxaliplatin-related excision repair cross-complementing 1 (ERCC1); biologic agent-related vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). Large-scale population analysis was performed to determine the association of gene expression with the clinicopathological features, in particular, the location of the colorectal cancer. From the results of our analysis of the mRNA expression of these 10 genes, we noted the strongest correlation between DPYD and TYMP, followed by TYMS and DHFR. The location of the colorectal cancer was classified into 4 regions (the right- and left-sided colon, rectosigmoid and rectum) and was compared with gene expression. A significant difference in all genes, apart from VEGF, was noted. Of the remaining 9 genes, the highest expression of TYMS and DPYD was observed in the right-sided colon; the highest expression of GGH and EGFR was noted in the left-sided colon; the highest expression of DHFR, FPGS, TOP1 and ERCC1 was noted in the rectosigmoid, whereas TYMP expression was approximately equivalent in the right-sided colon and rectum, and higher than that in other locations. The data generated from this study may prove to be useful for the development of individualized chemotherapeutic treatments for patients with colorectal cancer, and will mean that the tumor location is taken into account.

Purpose Gamma-glutamyl hydrolase ( GGH ), cyclin D1 ( CCND1 ) and thymidylate synthase ( TS ) genes encode enzymes that are involved in methotrexate (MTX) action. In a group of 184 RA patients treated with MTX, we have investigated whether selected polymorphisms in these genes modulate MTX efficacy and/or have impact on adverse drug effects (ADEs). Methods The efficacy of the MTX therapy has been estimated using the disease activity score in 28 joints (DAS28-ESR) based on EULAR criteria and relative DAS28 values (rDAS28). All adverse drug events were recorded. Patients were genotyped for selected polymorphisms of the GGH ( -354 G > T and 452 C > T ), CCND1 ( 870 A > G) and TYMS ( variable number of tandem repeats, VNTR, and G to C substitution of triple repeat, 3R allele) gene. Association studies have been performed between obtained genotypes and the efficacy and toxicity of MTX. Results According to the EULAR response criteria, 146 RA patients (79.3 %) were classified as responders (good/moderate response) and 38 (20.7 %) as non-responders (poor response). Higher frequency of the TYMS 3 G/3 G genotype has been found among non-responders as compared to individuals with remaining genotypes (p = 0.02). ADEs were recorded in 53 patients. Among those patients eight experienced bone marrow toxicity, all of them carried GGH -354GG genotype (p = 0.003). No other significant association were observed. Conclusion The 3 G/3 G genotype of the TYMS gene may indicate predisposition of poor response to MTX and GG genotype of GGH -354 T > G polymorphism may have high predictive value for myelosuppression in RA patients.

Purpose To investigate the correlation between common genetic polymorphisms of folylpolyglutamate synthase (FPGS), gamma-glutamyl hydrolase (GGH), and methylenetetrahydrofolate reductase (MTHFR) and serum levels of methotrexate (MTX) in Chinese children with acute lymphoblastic leukemia (ALL). Methods Ninety-one children with ALL who received high-dose MTX were recruited. The polymorphisms FPGS (rs1544105 G>A), GGH (rs3758149 C>T), and MTHFR (rs1801133 C>T) were genotyped through polymerase chain reaction-restriction fragment length polymorphism analysis. Serum MTX was measured by fluorescence polarization immunoassay. The association between targeted polymorphisms and MTX concentration-to-dose (C/D) ratios was assessed, and between targeted polymorphisms and the percent of MTX above the therapeutic threshold (40 µmol/L). Results The minor allele frequencies of rs1544105 G (34.1 %), rs3758149 T (19.2 %), and rs1801133 C (48.4 %) observed in our population were significantly lower than those reported for European populations (64.2, 30.8, and 69.0 %, respectively). The association between the GGH rs3758149 polymorphism and MTX C/D was gender-specific; in girls, the MTX C/D at 24 h of GGH rs3758149 CC carriers (12.09 μmol/L per g/m 2 ) was significantly lower than that of CT or TT carriers (16.80 μmol/L per g/m 2 ). The percent of serum MTX above the therapeutic threshold in GGH rs3758149 CC carriers (18.3 %) was significantly lower than that of CT and TT carriers (38.7 %). The MTX C/D ratios at 24 h and the percent of MTX >40 µmol/L for the A-T-T (three variant alleles) haplotype were significantly higher than those for other haplotypes combined ( P  < 0.05). Conclusions These data indicate that FPGS rs1544105, GGH rs3758149, and MTHFR rs1801133 polymorphisms contribute to the variability of MTX pharmacokinetics, and their genotyping may be useful to reduce toxicities associated with MTX therapy.

Repeated cycles of antibody-directed enzyme pro-drug therapy (ADEPT) and the use of glucarpidase in the detoxification of cytotoxic methotrexate (MTX) are highly desirable during cancer therapy but are hampered by the induced human antibody response to glucarpidase. Novel variants of glucarpidase (formal name: carboxypeptidase G2, CPG2) with epitopes not recognized by the immune system are likely to allow repeated cycles of ADEPT for effective cancer therapy. Towards this aim, over two thousand soil samples were collected and screened for folate hydrolyzing bacteria using folate as the sole carbon source. The work led to the isolation and the characterization of three new glucarpidase producing strains, which were designated as: Pseudomonas lubricans strain SF168, Stenotrophomonas sp SA and Xenophilus azovorans SN213. The CPG2 genes of Xenophilus azovorans SN213 (named Xen CPG2) and Stenotrophomonas sp SA (named Sten CPG2) were cloned and molecularly characterized. Both Xen CPG2 and Sten CPG2 share very close amino acid sequences (99%); we therefore, focused on the study of Xen CPG2. Finally, we demonstrated that a polyclonal antibody raised against our new CPG2, Xen CPG2, does not react with the CPG2 from Pseudomonas sp. strain RS-16 (Ps CPG2) that are currently in clinical use. The two enzymes, therefore could potentially be used consecutively in the ADEPT protocol to minimize the effect of the human antibody response that hampers current treatment with Ps CPG2. The identified novel CPG2 in this study will, therefore, pave the way for safer antibody directed enzyme pro-drug therapy for cancer treatment.

γ-Glutamyl hydrolase (GGH) regulates intracellular folates and antifolates such as methotrexate (MTX) for proper nucleotide biosynthesis and antifolate-induced cytotoxicity, respectively. In addition to genetic polymorphism and karyotypic abnormalities, methylation of CpG island 1 (CpG1) in the promoter region is found to modulate GGH activity by reducing GGH mRNA expression in acute lymphoblastic leukemia (ALL) cells. We aim to investigate methylation status of two CpG islands (CpG1 and CpG2) in the GGH promoter region in pediatric patients with ALL and acute myelogenous leukemia (AML). 70B-ALL, 29 AML, 10 ITP (idiopathic thrombocytopenic purpura) and 40 healthy children are recruited in the present study. MS-HRM (methylation-sensitive high-resolution melting) and bisulfite sequencing PCR (BSP) are used to detect methylation change and its level in CpG1 and CpG2 in the GGH promoter region. GGH mRNA expression is quantified by real-time PCR. Correlation between CpG island methylation and GGH mRNA expression is assessed by statistical software. Methylations of CpG1 are detected in leukemia cells samples obtained from 30.9% (21/68) of patients with ALL and 20.7% (6/29) of patients with AML. These methylations are not detected in the controls. Methylations of CpG2 are detected in leukemia cell samples obtained from 44.1% (30/68) of the ALL patients and 37.9% (11/29) of the AML patients. These percentages are significantly higher than that observed in the control cell samples: 6.0% (3/50) (Fisher's exact test, P = 0.000). The abundance of CpG1 methylation in all leukemia cell samples is classified as Grade I (methylation level is less than 10%) and the abundance of CpG2 methylation in leukemia cell samples is classified in separate grades. Our results indicate that methylation of CpG1 or hypermethylation (the methylation level is greater than 10%) of CpG2 could significantly reduce GGH mRNA expression in leukemia cells from the ALL and AML patients (ALL-CpG1: t = 4.632, P = 0.000; ALL-CpG2: t = 3.250, P = 0.006; AML-CpG1: t = -2.254, P = 0.037; AML-CpG2: t = 1.328, P = 0.202). Either methylation of CpG1 or hypermethylation of CpG2 in GGH promoter region can significantly reduce GGH mRNA expression in pediatric patients with acute leukemia, which can improve the response to treatment.

Hibernation is a state of extraordinary metabolic plasticity. The pathways of amino acid metabolism as they relate to nitrogen homeostasis in hibernating mammals in vivo are unknown. Here we show, using pulse isotopic tracing, evidence of increased myofibrillar (skeletal muscle) protein breakdown and suppressed whole-body production of metabolites in vivo throughout deep torpor. As whole-body production of metabolites is suppressed, amino acids with nitrogenous side chains accumulate during torpor, while urea cycle intermediates do not. Using 15 N stable isotope methodology in arctic ground squirrels ( Urocitellus parryii ), we provide evidence that free nitrogen is buffered and recycled into essential amino acids, non-essential amino acids and the gamma-glutamyl system during the inter-bout arousal period of hibernation. In the absence of nutrient intake or physical activity, our data illustrate the orchestration of metabolic pathways that sustain the provision of essential and non-essential amino acids and prevent ammonia toxicity during hibernation. Hibernation is a state of extreme metabolic and physiological plasticity occurring in conjunction with physical inactivity and prolonged fasting. Using in vivo tracing, Rice et al. reveal that nitrogen recycling staves off ammonia toxicity during hibernation in ground squirrels.