Kidney-type glutaminase (GLS1) plays a significant role in tumor metabolism. Our recent studies demonstrated that GLS1 was aberrantly expressed in hepatocellular carcinoma (HCC) and facilitated tumor progression. However, the roles of GLS1 in intrahepatic cholangiocarcinoma (ICC) remain largely unknown. Thus, the aim of this study was to evaluate the expression and clinical significance of GLS1 in ICC. For this purpose, combined data from the Oncomine database with those of immunohistochemistry were used to determine the expression levels of GLS1 in cancerous and non-cancerous tissues. Second, a wound-healing assay and Transwell assay were used to observe the effects of the knockdown and overexpression of GLS1 on the invasion and migration of ICC cells. We examined the associations between the expression of GLS1 and epithelial-mesenchymal transition (EMT)-related markers by western blot analysis. Finally, we examined the associations between GLS1 levels and clinicopathological factors or patient prognosis. The results revealed that GLS1 was overexpressed in different digestive system tumors, including ICC, and that GLS1 expression in ICC tissue was higher than that in peritumoral tissue. The overexpression of GLS1 in RBE cells induced metastasis and invasion. Moreover, the EMT-related markers, E-cadherin and Vimentin, were regulated by GLS1 in ICC cells. By contrast, the knockdown of GLS1 expression in QBC939 cells yielded opposite results. Clinically, a high expression of GLS1 in ICC samples negatively correlated with E-cadherin expression and positively correlated with Vimentin expression. GLS1 protein expression was associated with tumor differentiation (P=0.001) and lymphatic metastasis (P=0.029). Importantly, patients with a high GLS1 expression had a poorer overall survival (OS) and a shorter time to recurrence than patients with a low GLS1 expression. Multivariate analysis indicated that GLS1 expression was an independent prognostic indicator. On the whole, the findings of this study demonstrated that GLS1 is an independent prognostic biomarker of ICC. GLS1 facilitates ICC progression and may thus prove to be a therapeutic target in ICC.
Intrahepatic cholangiocarcinoma (ICC) is the second most common intrahepatic primary tumor after hepatocellular carcinoma (HCC) (
Glutaminase is an amidohydrolase that catalyzes the first step in the glutaminolysis of glutamine to glutamate. Glutaminase exists as two isoforms, GLS1 and GLS2, which were originally identified as kidney and liver glutaminases, respectively. The majority of cancer types, including ICC, require a constant supply of glutamine to support tumor progression and cell proliferation (
In this study, we examined the expression of GLS1 in ICC and investigated the role and mechanisms of action of GLS1 in ICC cell invasion and migration. In addition, clinical characteristics, such as overall survival (OS) and the cumulative recurrence rate were also assessed.
In this study, a tissue microarray was used containing 138 paired paraffin-embedded ICC tissue samples and corresponding peritumoral tissues samples obtained from patients who had undergone hepatic resection at Zhongshan Hospital, Fudan University from 2007 to 2012. The use of these tissue specimens was approved by the Zhongshan Hospital Research Ethics Committee and written consent was obtained from the patients. The detailed clinicopathological characteristics of the patients are presented in
Three ICC cell lines, QBC939, HCCC-9810 and RBE, were obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 µg/ml) (all Gibco; Thermo Fisher Scientific) under 95% air and 5% CO2 at 37°C.
A tissue microarray that included 138 ICC tissue samples with corresponding adjacent tissue samples was constructed by Shanghai Outdo Biotech Co., Ltd. (
For H&E staining, sections were stained with hematoxylin solution (0.2%) for 4 min, followed by eosin solution (0.5%) for 90 sec at room temperature.
Cell and tissue proteins were extracted using radioimmunoprecipitation assay buffer (cat. no. P0013C; Beyotime Institute of Biotechnology), and the protein concentration was measured using an enhanced Bicinchoninic Acid Protein Assay kit (cat. no. P0010; Beyotime Institute of Biotechnology). Proteins were loaded at 20 µg/lane, separated by 10% SDS-PAGE (cat. no. P0012A; Beyotime Institute of Biotechnology) and were transferred to polyvinylidene fluoride membranes (EMD Millipore) for western blot analysis. Subsequently, the membranes were blocked with TBS containing 0.1% Tween 20 and 5% non-fat milk for 2 h at room temperature, and were subsequently incubated with the following rabbit primary antibodies at 4°C for 12 h: Anti-GLS1 (1:1,000; cat. no. ab93434; Abcam), anti-E-cadherin (1:1,000; cat. no. 24E10; Cell Signaling Technology, Inc.), anti-Vimentin (1:500; cat. no. D21H3; Cell Signaling Technology, Inc.) and anti-GAPDH (1:1,000; cat. no. D16H11; Cell Signaling Technology, Inc.). The membranes were then rinsed and incubated with secondary antibody (1:5,000; cat. no. A0208; Beyotime Institute of Biotechnology) at room temperature for 2 h. Densitometric analysis using an enhanced chemiluminescence system (EMD Millipore) and ImageJ software (version 1.49; National Institutes of Health) was performed to detect protein expression.
siRNAs and a pcDNA plasmid that can regulate the human GLS1 gene were obtained from Shanghai Genomeditech Co. Two siRNAs were designed to silence GLS1, and the cells were randomly divided into the siRNA1-transfected cell group; siRNA2-transfected cell group; the negative control (NC) group, which was transfected with non-targeting siRNA; and the mock group, which consisted of untransfected cells. A pc-DNA3.1 plasmid was designed to induce overexpression of GLS1, and the cells were divided into the pc-DNA3.1-GLS1-transfected cell group; the NC group, which was transfected with an empty pc-DNA3.1 vector; and the mock group, which consisted of untransfected cells. The siRNA sequences were as follows: siRNA1, upstream 5′-CCAGGUUGAAAGAGUGUAUTT-3′, downstream 5′-AUACACUCUUUCAACCUGGTT-3′; siRNA2, upstream 5′-CCCUGAAGCAGUUCGAAAUTT-3′, downstream 5′AUUUCGAACUGCUUCAGGGTT-3′; NC, upstream 5′-UUCUCCGAACGUGUCACGUTT-3′, downstream 5′-ACGUGACACGUUCGGAGAATT-3′. Briefly, cells were seeded at a density of 1×106 cells/well in 6-well plates and were incubated until 70% confluence was reached. siRNAs and pc-DNA3.1 plasmids were transfected into the QBC939 and RBE cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 72 h, the cells were harvested for total protein extraction and examined by western blot analysis.
Cell migration was assessed using a wound-healing assay. ICC cells were plated in a 6-well plate and were incubated until they reached 100% confluence. Following serum starvation for 1 day, cells were wounded with a 200-µl plastic tip. Cells were washed three times with sterile PBS to remove the floating cells and were then incubated for 24 h at 37°C under 95% air and 5% CO2. The same wound areas were observed and images were captured under a light microscope (Olympus IX71; Olympus Corporation) at 0 and 24 h. The migratory abilities were quantified by measuring the percentage of the migration area of cells in the scratched regions, as follows: 0 h scratch area-24 h scratch area/0 h scratch area ×100%.
A Transwell assay was conducted to assess the invasive ability of cells in response to GLS1 overexpression or knockdown. The upper surface of the Transwell filter used in the assay was coated with Matrigel. Cells (1×105) suspended in 150 µl serum-free medium were added to the Transwell chamber (cat. no. 3413; Corning, Inc.), which was placed into a 24-well plate containing complete medium. After 24 h of incubation at 37°C, the filter was extracted and cells on the upper surface of the filter were removed with cotton swabs. Cells on the underside of the Transwell filter were fixed with 4% paraformaldehyde for 25 min and stained with 0.1% crystal violet for 15 min at 37°C, after which, images were captured (Olympus IX71; Olympus Corporation) and the number of cells was quantified.
Statistical analyses were performed using SPSS 23.0 (IBM Corp.) and GraphPad Prism7 (GraphPad Software, Inc.) software. The Student's t-test was used to compare differences between groups. One-way analysis of variance (ANOVA) was used to compare differences among groups. Spearman's correlation analysis was used for the correlation analysis and Fisher's exact test was used to determine the association of GLS1 with ICC characteristics. OS and the cumulative recurrence rate were determined using Kaplan-Meier survival curves and the log-rank test. Independent prognostic factors were evaluated by with Cox proportional hazards model. A P-value <0.05 was considered to indicate a statistically significant difference.
First, data from the Oncomine database were used to analyze GLS1 transcripts in 3 types of digestive system tumors. As shown in
To further examine the role of GLS1 in ICC cell invasion and migration, the GLS1 protein levels were examined in a panel of ICC cell lines (QBC939, HCCC-9810 and RBE) by western blot analysis (
Recently, a previous study demonstrated that GLS1 reduces cell-cell contact and increases cell motility by inducing EMT in lung cancer cells (
The samples were classified into 2 groups, a GLS1high (++, moderate; +++, strong) group and a GLS1low (−, absent; +, weak) group, according to the mean value of the expression of GLS1 in the tumor tissue samples (
In this study, we demonstrated that GLS1 was overexpressed in ICC tissue compared with adjacent normal tissue, and the downregulation of GLS1 expression in QBC939 cells suppressed ICC cell invasion and migration. The expression of the EMT mesenchymal marker, Vimentin, was downregulated following the knockdown of GLS1 expression in QBC939 cells. By contrast, the expression of the epithelial marker, E-cadherin, was upregulated. However, the overexpression of GLS1 in the RBE cells induced a lower expression of E-cadherin and a higher expression of Vimentin. Clinically, we detected GLS1 expression among 138 patients with ICC. The results revealed that a high GLS1 expression was strongly associated with poor tumor differentiation, lymphatic metastasis, early recurrence and an unfavorable prognosis. Patients with a high expression of GLS1 had a poorer OS and higher cumulative recurrence rates than patients with a low GLS1 expression.
The ‘Warburg effect’ describes the phenomenon of cancer cells creating energy predominantly from the glycolytic breakdown of glycose, rather than mitochondrial oxidative phosphorylation (
Previous studies have reported that EMT is a potential mechanism of cancer metastasis, and this process activates the mesenchymal phenotype and represses the epithelial phenotype, driving separation from the primary tumor (
In conclusion, the interactions of GLS1 with E-cadherin and Vimentin were confirmed in this study. However, whether other EMT markers are regulated by GLS1 warrants further investigation in the future. The results of this study, suggest that GLS1 may prove to be an innovative therapeutic target in patients with ICC.
Not applicable.
This study was supported by the Health research project in Jiangsu Province (grant no. H201661), Cancer Biology State Key Laboratory Project (grant no. CBSKL201717) and the National Natural Science Foundation of China (grant no. 81702861).
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
JC and DSB were involved in study design and drafted the manuscript. CZ, GQJ, SJJ and AWK were involved in TMA analysis. ZHG, DCY and QW were involved in statistical analysis. YQF, DWL and AQW were involved in clinical data collection. DSB was involved in the study design, financial support and proof-reading of the manuscript. All authors read and approved the final manuscript.
The use of these tissue specimens was approved by the Zhongshan Hospital Research Ethics Committee and written consent was obtained from the patients.
Not applicable.
The authors declare that they have no competing interests.
GLS1 expression is upregulated in primary ICC. (A) An Oncomine-based microarray database query of GLS1 gene expression in hepatocellular carcinoma, colorectal cancer and gastric cancer was performed. (B) The GLS1 protein level in 4 paired of human primary ICC tissue (T) and adjacent normal tissue (P) samples was determined by western blot analysis. (C and D) Immunohistochemistry was used to analyze the GLS1 expression in a TMA containing 138 samples of ICC patient tumor tissue and peritumoral tissue. Scale bars, 100 µm. *P<0.05, ***P<0.001. GLS1, kidney-type glutaminase; ICC, intrahepatic cholangiocarcinoma; TMA, tissue microarray.
GLS1 regulates invasion and migration in QBC939 and RBE cells. (A) Western blot analysis was used to analyze the expression of GLS1 in ICC cell lines. (B and C) ICC cells were transfected with siRNA/pcDNA, and western blot analysis was performed. (D and E) Effects of GLS1 expression on cell invasion and migration were examined by (D) a Transwell assay and (E) a wound-healing assay (E). *P<0.05, **P<0.01 and ***P<0.001. GLS1, kidney-type glutaminase; ICC, intrahepatic cholangiocarcinoma.
GLS1 successfully regulates EMT-related markers in QBC939 and RBE cells. (A and B) GLS1 regulated EMT-associated protein expression according to the results of western blot analysis. (C) Representative images of a sample from a patient with ICC that exhibited a GLS1high, Vimentinhigh and E-cadherinlow phenotype and a sample from a patient who exhibited a GLS1low, Vimentinlow and E-cadherinhigh phenotype. Scale bars, 100 µm. (D) GLS1 expression negatively correlated with E-cadherin expression and positively associ correlated ated with Vimentin expression. ***P<0.001. GLS1, kidney-type glutaminase; ICC, intrahepatic cholangiocarcinoma.
Log-rank tests and Kaplan-Meier survival curves of patients with ICC stratified according to their GLS1 protein expression. (A-D) Representative images of H&E and immunohistochemical staining for GLS1 expression. (A) Negative; (B) weak; (C) moderate; and (D) intensive. (E) OS curves stratified by GLS1 expression. (F) Cumulative recurrence rate curves stratified by GLS1 expression. Scale bars, 100 µm. GLS1, kidney-type glutaminase; ICC, intrahepatic cholangiocarcinoma; OS, overall survival.
Associations between GLS1 with clinicopathologic characteristics of the 138 patients with ICC.
No. of patients | |||
---|---|---|---|
Characteristic | GLS1low | GLS1high | P-value |
Sex | 0.609 | ||
Male | 25 | 33 | |
Female | 38 | 42 | |
Age, years | 0.090 | ||
≥53 | 27 | 43 | |
<53 | 36 | 32 | |
HBsAg | 0.657 | ||
Positive | 38 | 48 | |
Negative | 25 | 27 | |
Child-Pugh score | 0.299 | ||
A | 62 | 70 | |
B | 1 | 5 | |
Serum CA 19-9, ng/Ml | 0.061 | ||
≥37 | 33 | 51 | |
<37 | 30 | 24 | |
CEA | 0.215 | ||
≥3.4 | 22 | 34 | |
<3.4 | 41 | 41 | |
Serum ALT, U/l | 0.872 | ||
≥75 | 9 | 10 | |
<75 | 54 | 65 | |
Serum AFP, ng/ml | 0.871 | ||
≥20 | 7 | 9 | |
<20 | 56 | 66 | |
GGT | 0.485 | ||
≥75 | 29 | 39 | |
<75 | 34 | 36 | |
Cirrhosis | 0.586 | ||
Yes | 24 | 32 | |
No | 39 | 43 | |
Tumor size (diameter, cm) | 0.950 | ||
≥5 | 49 | 58 | |
<5 | 14 | 17 | |
Tumor number | 0.202 | ||
Multiple | 3 | 8 | |
Solitary | 60 | 67 | |
Embolus | 0.492 | ||
Yes | 9 | 14 | |
No | 54 | 61 | |
Capsulation | 0.252 | ||
Yes | 50 | 65 | |
No | 13 | 10 | |
Lymphatic metastasis | |||
Yes | 10 | 24 | |
No | 53 | 51 | |
Tumor differentiation | |||
III/IV | 21 | 46 | |
I/II | 42 | 29 |
Fisher's exact test was used to determine the P-values. GLS1high, ≥50% staining; GLS1low, <50% staining. Statistically significant values are shown in bold. AFP, α-fetoprotein; ALT, alanine aminotransferase; CA19-9, carbohydrate antigen 19-9; HBsag, hepatitis B surface antigen; GGT, γ-glutamyl transferase; ICC, intrahepatic cholangiocarcinoma; GLS1, kidney-type glutaminase.
Univariate and multivariate analyses of factors associated with recurrence and survival of patients with ICC.
Overall survival | Cumulative recurrence | |||||
---|---|---|---|---|---|---|
Univariate | Multivariate | Univariate | Multivariate | |||
Variable | P-value | HR (95% CI) | P-value | P-value | HR (95% CI) | P-value |
Sex (male vs. female) | 0.847 | NA | 0.921 | NA | ||
Age, years (≥53 vs. <53) | 0.491 | NA | 0.338 | NA | ||
HBsAg (positive vs negative) | 0.949 | NA | 0.518 | NA | ||
Child–Pugh score (a vs. B) | 0.131 | NA | 0.388 | NA | ||
Serum CA 19-9, ng/ml (≥37 vs., <37) | 0.272 | NA | 0.215 | NA | ||
Serum ALT, U/l (≥75 vs., <75) | 0.599 | NA | 0.799 | NA | ||
AFP | 0.130 | NA | 0.562 | NA | ||
CEA | 0.981 | NA | 0.361 | NA | ||
GGT | 0.044 | NS | 0.190 | NA | ||
Cirrhosis (yes vs. no) | 0.276 | NA | 0.458 | NA | ||
Tumor size (diameter, cm) (≥5 vs., <5) | 0.022 | 0.562 (0.338–0.935) | 0.026 | 0.005 | 0.540 (0.282–0.816) | 0.007 |
Tumor number (multiple vs. solitary) | 0.007 | NS | 0.001 | 0.479 (0.235–0.882) | 0.020 | |
Embolus (yes vs. no) | 0.032 | NS | 0.031 | 0.619 (0.403–0.952) | 0.290 | |
Capsulation (yes vs. no) | 0.252 | NA | 0.125 | NA | ||
Lymphatic metastasis (yes vs. no) | 0.007 | 0.539 (0.352–0.826) | 0.040 | 0.001 | 0.619 (0.352–0.826) | 0.029 |
Tumor differentiation (III/IV vs. I/II) | 0.069 | NS | 0.059 | NA | ||
GLS1 density (<50% vs. ≥50%) | <0.001 | 2.718 (1.820–4.059) | <0.001 | <0.001 | 2.774 (1.84–4.182) | <0.001 |
AFP, α-fetoprotein; ALT, alanine aminotransferase; CA19-9, carbohydrate antigen 19-9; HBsag, hepatitis B surface antigen; GGT, γ-glutamyl transferase; ICC, intrahepatic cholangiocarcinoma; GLS1, Kidney-type glutaminase; NS, not significant; NA, not available.