Regulation of stem-like cancer cells by glutamine through β-catenin pathway mediated by redox signaling
- Jianwei Liao†1, 2,
- Pan-Pan Liu†1,
- Guoxin Hou†1,
- Jiajia Shao†3,
- Jing Yang1,
- Kaiyan Liu1, 2,
- Wenhua Lu1,
- Shijun Wen1,
- Yumin Hu1 and
- Peng Huang1, 2Email author
© The Author(s). 2017
Received: 13 September 2016
Accepted: 23 February 2017
Published: 28 February 2017
Cancer stem cells (CSCs) are thought to play an important role in tumor recurrence and drug resistance, and present a major challenge in cancer therapy. The tumor microenvironment such as growth factors, nutrients and oxygen affect CSC generation and proliferation by providing the necessary energy sources and growth signals. The side population (SP) analysis has been used to detect the stem-like cancer cell populations based on their high expression of ABCG2 that exports Hoechst-33342 and certain cytotoxic drugs from the cells. The purpose of this research is to investigate the effect of a main nutrient molecule, glutamine, on SP cells and the possible underlying mechanism(s).
Biochemical assays and flow cytometric analysis were used to evaluate the effect of glutamine on stem-like side population cells in vitro. Molecular analyses including RNAi interfering, qRT-PCR, and immunoblotting were employed to investigate the molecular signaling in response to glutamine deprivation and its influence on tumor formation capacity in vivo.
We show that glutamine supports the maintenance of the stem cell phenotype by promoting glutathione synthesis and thus maintaining redox balance for SP cells. A deprivation of glutamine in the culture medium significantly reduced the proportion of SP cells. L-asparaginase, an enzyme that catalyzes the hydrolysis of asparagine and glutamine to aspartic acid and glutamate, respectively, mimics the effect of glutamine withdrawal and also diminished the proportion of SP cells. Mechanistically, glutamine deprivation increases intracellular ROS levels, leading to down-regulation of the β-catenin pathway.
Glutamine plays a significant role in maintaining the stemness of cancer cells by a redox-mediated mechanism mediated by β-catenin. Inhibition of glutamine metabolism or deprivation of glutamine by L-asparaginase may be a new strategy to eliminate CSCs and overcome drug resistance.
KeywordsGlutamine Side population Cancer stem cell L-asparaginase Sox2 ROS β-catenin
Despite major progress made in our understanding of basic cancer biology and new therapeutic targets in the past decades, the clinical outcomes for certain types of cancers such as lung, liver, and pancreatic cancers remain unsatisfactory. Extensive studies have indicated that cancer stem cells (CSCs) may play a key role in tumor initiation and disease recurrence [1–5], but finding effective measure to eradicate CSCs still remain as a major challenge. Recent advancement in high throughput screening technology has enabled the identification of salinomycin as a selective toxic agent against cancer stem cells . Furthermore, the self-renewal properties of cancer stem cells and the signals from their microenvironment may also be used to preferentially target CSCs. Indeed, the critical role of certain cytokines, pH, and oxygen in affecting CSCs proliferation and differentiation have been evaluated [7, 8]. However, the impact of nutrients in the tumor microenvironment on the CSCs remains largely unknown.
Cancer cells demand rapid ATP production to maintain their active cellular processes, require active biosynthesis of macromolecules to support cell division, and need a tightly controlled ROS metabolism to maintain cellular redox balance and cell survival . Glucose and glutamine are the two major nutrients whose metabolism is often altered in cancer cells. The best characterized metabolic shift in tumor cells is the Warburg effect, which refers to the higher aerobic glycolysis observed in the majority of cancer cells compared to normal cells . Notably, recent studies suggest that CSCs seem to have higher glycolytic activity and lower mitochondria respiration compared to the bulk of “regular” cancer cells [11–13]. Glucose in tumor microenvironment induces a reversible increase of stem-like side population cells . The glucose-driven glycolysis also plays key roles in maintaining hematopoietic stem cells (HSCs) and controlling differentiation, and HSCs exhibit high glycolysis and low oxidative phosphorylation associated with decreased mitochondrial mass and mutations of certain mitochondrial gene . Thus, it is not surprising that glucose in the tumor microenvironment plays an important role in the regulation of stem cell (29).
Glucose and glutamine metabolisms are interrelated at multiple levels. Glutamine transport is the rate-limiting step in the activation of mTOR signaling pathway, and this latter event induces glucose uptake through upregulation of the glucose transporter Glut1 [15, 16]. Glucose and glutamine are both precursors of the tricarboxylic acid (TCA) cycle as well as precursors of the lipids production, nucleotides and amino acids synthesis . However, the effect of glutamine on regulation of CSCs is largely unknown. In this study, we used side population (SP) cells as our in vitro model to study the potential impact of glutamine on stem-like cancer cells. Glutamine depletion from the culture medium resulted in a decrease in SP subpopulation in vitro. We also found that the expression of several key stem cell associated markers (i.e. Sox2 and ABCG2) were also down-regulated upon glutamine deprivation by multiple methods. Moreover, glutamine deprivation led to an increase of reactive oxygen species (ROS), which in turn negatively regulated β-catenin pathway to decrease the fraction of SP cells. Finally, we investigated the potential role of glutamine deprivation and L-asparaginase on A549 cells tumorigenicity capacity in vivo.
Chemicals and reagents
Hoechst 33342, verapamil, glutaminase, L-asparaginase, and 3-Amino-1,2,4-triazole (ATZ), 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), hydroethidine, Rhodamine 123 were purchased from Sigma (St Louis, MO, USA). Rabbit monoclonal anti-Axin2 (D48G4), rabbit monoclonal anti-Survivin (71G4B7), rabbit polyclonal anti-phospho-β-catenin (Ser33/37/Thr41), rabbit monoclonal anti-phospho-Akt (Ser473) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal anti-c-Myc (9E10) was purchased from Santa Cruz (Santa Cruz, CA, USA). Rabbit polyclonal anti-CyclinD1 antibody was obtained from GeneTex (San Antonio, TX, USA). Mouse monoclonal anti-β-catenin (C47H1), rabbit monoclonal anti-Sox-2, rabbit monoclonal anti-ABCG2, and mouse monoclonal anti-β-actin antibodies were purchased from Abcam (Cambridge, UK). CM-DCFDA, Lipofetamine RNAiMAX and Opti-MEM were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA).
Cells and cell cultures
Human non-small cell lung carcinoma (NSCLC) A549 and pancreatic cancer AsPC-1 cells were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA) and routinely maintained in RPMI 1640, supplemented with 10% fetal bovine serum (Invitrogen Life Technologies). Glioblastoma cancer stem cell lines GSC11 and GSC23 originally derived from human glioblastoma tissues were maintained in DMEM/F-12 (Hyclone) supplemented with B-27 (Invitrogen), 2 mM glutamine (Mediatech), 20 ng/ml recombinant human epidermal growth factor (EGF; R&D Systems), and 20 ng/ml basic fibroblast growth factor (bFGF; R&D Systems) as described previously . All cell lines were incubated at 37 °C in a humidified atmosphere with 5% CO2.
Measurement of intracellular ATP
Cellular ATP levels were determined using the ATP-based CellTiter-Glo luminescent Cell Viability kit (Promega, Madison, USA) according to the manufacturer’s instructions with the following modifications. Briefly, Cells were plated in triplicate in 96-well plates to allow for attachment overnight, and then the culture was switched to glutamine-free medium or L-asparaginase (L-ASP) was added to the culture for different times to deplete glutamine. The cell samples were then mixed with equal volume of the single-step reagent provided with the ATP-based CellTiter-Glo kit and rocked for 2 min followed by incubation at room temperature for 15 min. Then luminescence levels were measured using a luminescent plate reader (Thermo Fisher Varioskan Flash; Waltham, MA).
Flow cytometry analysis of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP)
Intracellular ROS (H2O2) contents were measured by incubating cells with 10 μM CM-DCFDA at 37 °C for 1 h followed by detection using flow cytometry (Beckman Coulter). Intracellular superoxide level was measured by incubating cells with 50 ng/ml Het at 37 °C for 30 min before detection by flow cytometry. MMP was detected after incubation of cells with 1 μM rhodamine-123 for 30 min flowed by flow cytometry analysis.
Measurement of cellular glutathione
Cellular glutathione (GSH) concentrations were measured using the GSH-Glo Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol with the following modifications. Briefly, cells were seeded in 96-well plates and incubated with either complete medium or glutamine-free medium for 24 h, 48 h, or 72 h. The culture medium was then removed and the cells were lysed with 100 μl reaction buffer provided in the kit. After incubation for 30 min, 100 μl detection buffer was added and incubated for another 15 min at room temperature. GSH contents were measured using a luminescent plate reader, and were normalized by cell numbers.
Determination of NADP+/NADPH
NADP+, NADPH, and their ratio were measured using the NADP/NADPH Quantitation Colorimetric Kit (BioVision Inc., Milpitas, CA, USA). Briefly, after A549 cells were cultured with or without glutamine/L-Asparaginase for 48–72 h, the cells were washed with cold PBS and then lysed using NADP/NADPH extraction buffer on ice for 10 min. The cell lysates were spun down and the supernatants were used for measurement of NADP+/NADPH using the assay conditions recommended by the manufacturer (BioVision Inc.).
RNA extraction and quantitative real-time PCR analysis
Total RNA was extracted with TRIZOL (Ambion, Austin, TX, USA) from A549 cells after cultured in RPMI 1640 with or without glutamine, L-asparaginase or H2O2. cDNA was generated from equal amount of total RNA (1 μg) using Prime Script RT reagent kit with DNA Eraser (Takara Biotechnology, Dalian, Liaoning, China). Specific primers used for the amplification of indicated genes were listed in Additional file 1: Table S1 and S2. Real-time PCR was performed using SYBR Premix Ex Taq II kit (TliRNase H Plus, Takara Biotechnology, Dalian, Liaoning, China) and the CFX96 real-time system (Bio-Rad Laboratories, Hercules, CA, USA). The RT-PCR amplification reaction program consisted of one cycle of 95 °C/30S and 40 cycles of 95 °C/5S → 60 °C/30S. β-actin was used as an internal control for normalization.
Protein extraction and western blot analysis
A modified RIPA buffer (150 mM NaCl, 50 mM Tris, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM EDTA) with a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Roche, Indianapolis, Indiana, USA) was used for protein isolation. Cells were washed twice with ice-cold PBS and lysed in 100–200 μl RIPA buffer for 30 min. Cell debris was removed by centrifugation at 12,000 rpm for 15 min at 4 °C. The supernatants were collected, and protein concentrations were determined using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). An equal quantity of proteins from each experimental condition were subjected to electrophoresis in denaturing 10% SDS-polyacrylamide gel, and then transferred to a PVDF membrane, which was probed for p-β-catenin, β-catenin, p-Akt, ABCG2, SOX-2 and β-actin here using as internal control.
RNA interference assay
Small RNA interference (siRNA) for knockdown of β-catenin expression in A549 cells was performed using Lipofetamine RNAiMAX Reagent. Briefly, 2x105 A549 cells per well were plated in six-well plates. After overnight incubation, the culture medium in each well was replaced with 2 ml fresh medium containing 250 μl transfection reagents (containing Opti-MEM, β-catenin siRNA or scrambled RNA, and Lipofetamine RNAiMAX). After incubation for 48 h, the transiently transfected cells were collected and RNA was extracted for analysis by qRT-PCR.
Side population analysis
Cells were washed with PBS, trypsinized, and re-suspended in pre-warmed RPMI 1640 medium containing 2% FBS with or without glutamine at a final density of 1x106 cells/ml. Cell staining was performed according to the method described by Goodell et al.  with the following modifications. Briefly, the cells were incubated with Hoechst 33342 (5 μg/ml) in the presence or absence of the ABC transporter inhibitor verapamil (50 μM) for 90 min at 37 °C in dark with intermittent shaking. The cells were then washed and re-suspended in cold PBS. Single cell suspension was obtained using a 70 μm cell strainer. Cells were kept at 4 °C for flow cytometry analysis or sorting on a MoFlo XDP Cell Sorter (Beckman Coulter).
Colony formation and tumor cell sphere forming assays
A549 cells were seeded in six-well plates at a density of 400 cells per well and cultured at 37 °C for two weeks. At the end of the incubation, the cells were fixed with 100% methanol and stained with 0.1% (w/v) Crystal Violet, and the colonies were counted. Each measurement was performed in triplicate and the experiments were each performed at least three times. For neurosphere formation assay, glioblastoma stem cells GSC11 and GSC23 were seeded in 6-well plates in a range of 100–1000 cells per well, cultured in the indicated medium with or without glutamine for 2 weeks, and then the cell spheres were examined under a light microscope (Nikon).
Evaluation of in vivo tumorigenicity
To test the effect of glutamine deprivation on tumor-initiating capacity, A549 cells were treated under glutamine deprived conditions for 5 days in vitro. The cells were then harvested and inoculated subcutaneously into the flanks of athymic nude mice with the indicated cell numbers per injection site. The presence or absence of a visible tumor was evaluated and tumor growth was monitored every 3 days. The mice were sacrificed by the end of two months or when the tumors reached a maximum size of 1,000 mm3. Tumor volume was calculated by the formula 0.5 × length × width2. All animal experiments were conducted in accordance with the institutional guidelines and approved by the Animal Care and Use Committee of Sun Yat-sen University Cancer Center.
Data were analyzed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA). Data are presented by error bars (mean +/− SD) from experiments in triplicate unless otherwise noted. A two tailed Student’s t test was used to determine the statistical significance of difference between samples.
Glutamine deprivation reduced stem-like SP cells
Impact of glutamine on clonogenic capacity and expression of stem cell markers
To test if the impact of glutamine deprivation on SP cells could be reversed by replenishment of glutamine, A549 cells were first cultured in glutamine-free medium for 48 h to induce a decrease of SP cells. The cells were then switched to glutamine-containing medium for another 48 h, and SP cells were measured. The results showed that there was a substantial recovery of SP population after 48 h in glutamine-replenished medium (Additional file 1: Figure S4A), accompanied by corresponding changes in the expression of stem cell markers including ABCG2, ALDH1, SOX2, and CD44 (Additional file 1: Figure S4B). These data suggest that the effect of glutamine on stemness was reversible.
Glutamine deprivation increased ROS levels through attenuation of the GSH antioxidant system
Glutamine regulated the proportion of SP cells through ROS/beta-catenin pathway
To further test the role of β-catenin in regulating stem cells, we used siRNA to suppress the expression of beta-catenin, and evaluated its impact on stemness. As shown in Fig. 6c and d, siRNA effectively suppressed the expression of β-catenin, leading to a significant decrease of ABCG2 expression. These data together suggested that glutamine regulated the proportion of stem-like side population cells might be at least in part through ROS-mediated β-catenin phosphorylation, which leads to β-catenin protein degradation by proteasome .
Effect of glutamine on the ability of cancer cells to form tumor in vivo
Recent studies suggest that the microenvironment in the stem cell niches plays a major role in regulation of stemness, and promotes the long-term survival and self-renew of CSCs [7, 39–42]. Among the nutrients in the tumor microenvironment, glutamine is an important amino acid on which many cancer cells rely for survival and proliferation. In fact, addiction to glutamine is often observed in cancer cells, which use this amino acid as an energy source, a metabolic intermediate for synthesis of other biomolecules, and a precursor for synthesis of glutathione to maintain redox balance [17, 43–45]. Although the reasons for cancer cell dependency on glutamine are not entirely clear, the high demand of energy (ATP) and metabolic intermediates for active cell growth and the increased need for glutathione to counteract ROS stress under oncogenic signals are among the possible explanations for glutamine addiction. Wang et al. revealed that ASCT2 is important for melanoma and inhibition of this glutamine transporter could suppress cell proliferation . In this study, we discovered another important role of glutamine in maintaining stem-like cancer cells, using side population in lung cancer as an experimental model system.
Our study showed that glutamine deprivation induced a significant decrease in SP cells associated with a down regulation of ABCG2 and Sox-2. Interestingly, the rate of decrease in SP cells induced by glutamine depletion was much slower than that induced by glucose deprivation, suggesting that these two major nutrients seem to affect cancer stem cells by different mechanisms. In fact, it has been shown that glucose affects CSCs through a mechanism involving Akt-mediated regulation of ABCG2 expression (29), whereas the current study showed that the Akt activation status seemed not associated with the changes in SP cells induced by glutamine (Additional file 1: Figure S4B). The results of our study suggest that a mechanism by which glutamine affects SP cells may be through ROS-mediated activation of the β-catenin pathway, which regulates the expression of certain stem cell-related genes . Recent studies suggest that CSCs seem to have higher glycolytic activity and may be more dependent on glucose to generate ATP compared to the bulk of general cancer cells [11–13]. Thus, glucose deprivation could cause a severe energy deficiency in CSCs, leading to their rapid decrease. In contrast, a major role of glutamine in CSCs is to function as a metabolic precursor for the synthesis of glutathione to maintain redox balance and keep the intracellular ROS at a relative low level. Thus, a depletion of glutamine would cause an increase of ROS, which tend to induce cell differentiation and eventually lead to a gradual decrease in CSC population. These different roles of glucose and glutamine in energy metabolism and redox homeostasis may explain their different dynamics in impacting CSCs.
In our study, glutamine deprivation caused an increase in β-catenin phosphorylation, leading to its inactivation and a decreased expression of its down-stream targets survivin and Axin2. These results suggest that the β-catenin pathway might play an important role in mediating the down-regulation of SP cells induced by glutamine depletion, which led to an increase in ROS. It is known that ROS negatively regulates β-catenin . Interestingly, a previous study showed that blocking glutamine metabolism could inhibit cancer metastasis , which is a property of cancer stem cells. Consistently, we found that glutamine deprivation could induce a decrease of MMP7 (data not shown), a marker of cancer metastasis and also a downstream target of β-catenin.
Cancer stem cells in general are slow-cycling or quiescent cells that retain BrdU-labeling over a long period due to slow division [49, 50], which render them less sensitive to many chemotherapeutic agents that target fast-proliferating cells. A low level of intracellular ROS seems critical to maintain the quiescent status of cancer cells . To maintain a low ROS level, CSCs require high capacity of antioxidants to counteract ROS generated during active cellular metabolism. Indeed, two important transcription factors, FoxO and P53, have been shown to play a significant role in regulation of cellular ROS, and both are considered to be important molecules for the maintenance of stem cells [52, 53]. The reduced form of glutathione (GSH) is a highly abundant antioxidant in the cells, and plays an important role in keeping redox balance and promoting cell viability and drug resistance. In fact, cancer cells with positive CD44, which interact with the cysteine transporter xCT and promote GSH synthesis , exhibit grow advantage and resistant to certain therapy [55, 56].
Stem–like side population cells are more addicted to glutamine. Deprivation of glutamine can decrease the fraction of SP cells and stem cell markers (SOX-2 and ABCG2). Glutamine deprivation increases cellular ROS through attenuating glutathione synthesis, while increased ROS suppresses β-catenin pathway through inducing its phosphorylation and degradation. Tumor formation capacity in vivo was weakened through blocking glutamine utility by L-asparaginase.
ATP-Binding Cassette, Sub-Family G (WHITE), member 2
Acute lymphoblastic leukemia
ASC amino-acid transporter 2
Cancer stem cell
Forkhead box O
Glioblastoma stem cell
- H2O2 :
Mitochondrial membrane potential
3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
Nicotinamide adenine dinucleotide phosphate
Reactive oxygen species
SRY (sex determining region Y)-box 2
- TCA cycle:
Tricarboxylic acid cycle
The authors thank Dr. Helene Pelicano for helpful discussion and comments.
This work was supported in part by grants from the National Natural Science Foundation of China (81430060 and 81502573), Guangzhou Innovative Research Program (LCY201317), Guangzhou Medicare Collaborative Innovation Program (210508020250), and from Natural Science Foundation of Guangdong Province (2014A030310421).
Availability data and materials
Please contact author for data requests.
JL, P-PL, GH, JS performed experiments, analyzed data, and wrote the manuscript; JY, KL performed experiments and analyzed data; WL performed animal study; SW, YH participated in research design and data analysis; PH designed the study, analyzed data, and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
The animal study reported in this manuscript was performed in compliance with the protocol and procedures approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University Cancer Center.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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