Journal of Molecular Medicine https://doi.org/10.1007/s00109-018-1659-0

ORIGINAL ARTICLE

Targeting glutaminase-mediated glutamine dependence in papillary thyroid cancer Yang Yu 1 & Xiaohui Yu 1 & Chenling Fan 1 & Hong Wang 1 & Renee Wang 1 & Chen Feng 2 & Haixia Guan 1 Received: 17 November 2017 / Revised: 4 June 2018 / Accepted: 5 June 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Papillary thyroid cancer is a prevalent endocrine malignancy. Although alterations in glutamine metabolism have been reported in several types of hematological and solid tumors, little is known about the functions of glutamine and glutaminolysis-associated proteins in papillary thyroid cancer. Here, we demonstrated the glutamine dependence of papillary thyroid cancer cells, and with the use of RT2-PCR arrays, we screened for the aberrant overexpression of glutaminase in human papillary thyroid cancer tissues and cells. These results were later confirmed via real-time PCR, Western blots, and immunohistochemical staining. We found that the levels of glutaminase were significantly correlated with extrathyroidal extension. Inhibition of GLS suppressed glutaminolysis and reduced mitochondrial respiration. The proliferative, viable, migratory, and invasive abilities of papillary thyroid cancer cells were impaired by both the pharmacological inhibition and the genetic knockdown of glutaminase. Additionally, the inhibition of glutaminase deactivated the mechanistic target of the rapamycin complex 1 (mTORC1) signaling pathway, promoting autophagy and apoptosis. Collectively, these findings show that glutaminase-mediated glutamine dependence may be a potential therapeutic target for papillary thyroid cancer. Key messages & PTC cells are glutamine-dependent, and GLS is aberrantly overexpressed in PTC. & Inhibition of GLS suppressed glutaminolysis and reduced mitochondrial respiration. & Inhibition of GLS impairs the viability of PTC cells. & GLS blockade causes deactivation of mTORC1 and induction of autophagy and apoptosis. & GLS may be a potential therapeutic target for PTC. Keywords Glutaminase . Glutamine . Glutaminolysis . Papillary thyroid cancer

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00109-018-1659-0) contains supplementary material, which is available to authorized users. * Chen Feng [email protected] * Haixia Guan [email protected] 1

Department of Endocrinology and Metabolism, Institute of Endocrinology, Liaoning Provincial Key Laboratory of Endocrine Diseases, The First Affiliated Hospital of China Medical University, Shenyang 110001, China

2

Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001, China

Introduction Thyroid cancer is one of the most common endocrine malignancies, with approximately 62,000 new cases and 1950 deaths in 2015 [1]. Papillary thyroid cancer (PTC) is the predominant histological type of thyroid cancer, with an average annual percent change of incidence rate of 6.26% [2]. Although PTC is curable with a relatively good prognosis, the incidence-based mortality rate has increased significantly [3]. In addition, recurrence and death from PTC can occur even 30 years after a patient undergoes treatment [4]. As a result, novel biomarkers that regulate PTC progression and advancement may serve as important therapeutic targets.

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Altered cell metabolism is believed to be a hallmark of cancer [5]. Increased aerobic glycolysis (also known as the Warburg effect) and glutaminolysis have been verified in many malignancies [6, 7]. Although glutamine is a non-essential amino acid, it is the most abundant amino acid in the human bloodstream. Glutamine is essential for the viability of several cancer cell types [8–10]. Several selective amino acid transporters on the plasma membrane facilitate the entry of glutamine into cells [11], and glutamine is the major carbon source and nitrogen d o n o r f o r b i o e n e rg et i c s a n d b i o s y n t h e s i s [ 1 0 ] . Glutaminolysis begins with the conversion of glutamine to glutamate, which is catalyzed by mitochondrial glutaminase (GLS). GLS exists as two major splice variants, the longer form kidney-type glutaminase (KGA) and the shorter form glutaminase C (GAC) [12–14]. Glutamate is then converted to α-ketoglutarate by either glutamate dehydrogenase (GDH) or transaminase [15]. Glutaminolysis contributes to the biosynthesis of proteins, lipids, nucleotides, glucosamine-6-phosphate, non-essential amino acids, and ATP as well as regulating redox homeostasis [16]. In addition, glutamine plays a crucial role in the uptake of certain essential amino acids, including leucine, isoleucine, valine, methionine, and tyrosine, for which it is exchanged by associated transporters [17]. In proliferating cancer cells, glutamine activates major signaling pathways and serves as a vital source for the replenishment of TCA intermediates (anaplerosis) to sustain cell survival and proliferation [18–20]. Recently, it was verified that glutamine metabolism is altered in several types of hematological and solid tumors, which could be used as a therapeutic biomarker for many malignancies [21–24]. However, altered glutamine metabolism in thyroid cancer is poorly investigated. In 2016, Kim et al. reported altered expression levels of glutamine metabolism-related proteins in various histological subtypes of thyroid cancer [25]. Unfortunately, the specific function of glutamine-related proteins in thyroid cancer remains unclear. In this study, we confirmed that PTC cells were glutamine-sensitive and glutamine-dependent. The aberrant overexpression of GLS in PTC was screened using RT2-PCR arrays, and it was confirmed in fresh PTC and matched non-tumor thyroid tissues as well as in four different human PTC cell lines. We further characterized GLS in PTC cell lines using pharmacological inhibitors and specific siRNA transfection. The inhibition of GLS significantly decreased the proliferation, migration, and invasion abilities of PTC cell lines by deactivating the mechanistic target of the rapamycin complex 1 (mTORC1) signaling pathway and by promoting autophagy and apoptosis. Our study provides the first evidence that GLS may represent a novel biomarker for PTC treatment.

Materials and methods Human thyroid tissue samples PTC tissues and non-tumor thyroid tissues were obtained from patients who underwent thyroidectomy and were histopathologically diagnosed with PTC at the First Affiliated Hospital of China Medical University from 2008 to 2017. Patients who underwent secondary surgeries or those diagnosed with other histological thyroid cancers were excluded. The non-tumor tissues were obtained from surrounding tissues at least 1 cm away from the tumor. TNM classification was assessed according to the American Joint Committee on Cancer (7th edition). This study was supported by the First Affiliated Hospital of China Medical University and was conducted in accordance with the Declaration of Helsinki. Tissue specimens were collected with patients’ informed consent according to institutional ethical guidelines approved by the Institute Research Ethics Committee.

Human thyroid cell lines and reagents We obtained four different human PTC cell lines (K1, IHH4, BCPAP, and TPC-1) and a noncancerous thyroid cell line (Nthy-ori 3-1). K1 and BCPAP were purchased from the Health Protection Agency Culture Collections (Salisbury, UK) and the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures; Braunschweig, Germany), respectively. IHH4 cells were purchased from the Japanese Collection of Research Bioresources (Japan). TPC-1 cells were a kind gift from Dr. Bryan R. Haugen (CO, USA). Nthy-ori 3-1 cells were immortalized with SV40 large T antigen and were purchased from the same organization as K1. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (TPC-1; Gibco, Thermo Fisher Scientific, USA), RPMI-1640 (BCPAP, K1, and Nthy-ori 3-1; Gibco), or DMEM/RPMI1640 (50/50 mixture; IHH4) supplemented with 10% fetal bovine serum (FBS) and 5% CO2 at 37 °C. To determine the effects of bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2yl)ethyl sulfide (BPTES; Sigma-Aldrich) and CB-839 (Selleck, America) on PTC cell lines, cells were grown for 48 h in DMEM, RPMI-1640, or DMEM/RPMI-1640 with glutamine in the presence of 10 μM BPTES or 1 μM CB-839 in 0.1% DMSO or vehicle (0.1% DMSO). The identity of the cultured cell lines was confirmed with short tandem repeat profiling.

Glutamine deprivation assays Glutamine deprivation assays were conducted at 37 °C under 5% CO2 with commercial cell culture media containing no

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glutamine, including DMEM (TPC-1; Gibco) and RPMI1640 (BCPAP, K1, and Nthy-ori 3-1; Gibco) supplemented with 10% FBS. For IHH4 cells, a 50/50 mixture of DMEM and RPMI-1640 was used. For more convenient comparisons, glucose (Sigma-Aldrich) was added to all culture media to a final concentration of 25 mM, and glutamine (Sigma-Aldrich) was added to the culture media of control cells to a final concentration of 4 mM.

RT2 profiler PCR arrays Total RNA was extracted from thyroid tissues and cells with an RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. The expression of genes with specific roles in regulating amino acid metabolism was examined using custom SYBR Green-based RT2 PCR arrays (Qiagen).

Tissue microarrays and immunohistochemistry Tissue microarrays were constructed with 54 pairs of PTC tissues (PTC tissues and matched non-tumor tissues from the same patient) and an excess of 91 PTC tissues and 76 nontumor thyroid tissues. Paraffin-embedded tissue blocks were cut into 4-μm sections, and immunohistochemistry (IHC) staining was performed. After being incubated with a primary antibody against GLS (Abcam, Cambridge, UK) overnight at 4 °C, the sections were incubated with the Polink-1 horseradish peroxidase (HRP) DAB Detection System (ZS Bio, China), and a color reaction was performed with 3,3′-diaminobenzidine (DAB; ZS Bio). For the negative control, sections were incubated in phosphate-buffered saline (PBS) instead of primary antibody. Normal human kidney tissues were stained as a positive control to confirm the specificity of the antibody according to the manufacturers’ protocols. All sections were counterstained with hematoxylin.

Interpretation of IHC results The expression of GLS was assessed as previously described [26]. The staining results were scored by two experienced pathologists blinded to the clinical factors, and disagreements were discussed and resolved by consensus. Staining intensity was classified using the following (point) system: (0) absent, (1) weak, (2) intermediate, and (3) strong. Staining extent was assessed by the percentage of positive staining tumor cells using the following (point) system: (0) < 5%, (1) 5–25%, (2) 26–50%, (3) 51–75%, and (4) > 75%. The staining results were evaluated by multiplying the staining intensity and scope points. The resulting final immunoreactive score (IRS) was classified into four categories: < 4, 4, 6, or ≥ 8, which indicated negative (−), weak (+), medium (++), and strong (+++) staining, respectively. The former two categories were

assigned to GLS-negative groups, and the latter two were assigned to GLS-positive groups.

Transfection Cells were transfected using Lipofectamine 2000 Reagent (Invitrogen; Carlsbad, CA, USA) according to the manufacturer’s protocol. Briefly, 2 × 105 cells/well were seeded onto six-well plates and were incubated for 24 h to 70–80% confluence. Cells were transfected with either a GLS-targeting siRNA or a negative control (GenePharma, Shanghai, China). The transfection efficacy was assessed by Western blots.

Cell proliferation and viability assay Cell proliferation and viability were assessed with Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan), trypan blue staining, and the CellTiter-Lumi™ Luminescent Cell Viability Assay (CTL, Beyotime Biotechnology, China) according to the manufacturers’ protocols. For the CCK-8 assay, cells were seeded onto 96-well plates, and the optical density (OD) was measured for 3 days with a microplate reader (Tecan i-control, Sweden) at 450 nm. For trypan blue staining, 80,000–160,000 cells were seeded onto 24-well plates and counted with a TC20 Automated Cell Counter (Bio-Rad, California, USA) after 24, 48, and 72 h. For the ATP-dependent CellTiter-Lumi™ Luminescent Cell Viability Assay, 2000–3000 cells/100 μL were seeded onto 96-well white cell culture plates. After 48 h, 100 μL of CTL reagent was added to each well, and luminescence was read with a microplate reader.

Metabolic assays The intracellular levels of α-KG were determined with a commercially available kit (Biovision, USA). Briefly, 2 × 106 cells were rapidly homogenized in 100 μL of α-KG assay buffer, and the supernatant was deproteinized using 10-kDa molecular weight Nanosep® Centrifugal Devices (Pall Life Sciences, USA). The measurement of α-KG and background correction were performed according to the manufacturer’s instructions. The intracellular levels of glutamate were measured using a glutamate detection kit (Nanjing Jiancheng Bioengineering Institute, China) with a spectrophotometer at 340 nm (UV-2550, Shimadzu, Japan) following the manufacturer’s protocol. Oxygen consumption was measured with the XF Cell Mito Stress Test Kit using a Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA) according to the manufacturer’s instructions. 1.2 × 104 cells/well (K1) or 4 × 104 cells/well (TPC-1) were seeded onto an assay microplate and incubated overnight. Assay compounds were added sequentially at the

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following final concentrations: oligomycin (K1, 1 μM; TPC1, 2 μM), FCCP (0.5 μM), rotenone and antimycin A (0.5 μM). Oxygen consumption rate (OCR) values were normalized with cell protein estimation (BCA protein assay kit, Beyotime Biotechnology, China).

Cell migration and invasion assays Cell migration and invasion was evaluated by Transwell cell migration assay and Matrigel invasion assay, respectively. Cells cultured in media without FBS were seeded into the upper compartment with or without Matrigel (BD Biosciences, San Jose, CA, USA) and were incubated at 37 °C with 5% CO2. Cells that adhered to the surface of the lower compartment were counted by calculating the average number in ten random visual fields.

RNA isolation and real-time PCR Total RNA was isolated with TRIzol reagent (Takara Biotechnology, Dalian, China) according to the manufacturer’s protocol and was processed to cDNA with a reverse transcription kit (Takara Biotechnology). Real-time quantitative RT-PCR was performed using the LightCycler 480 II Real-Time PCR system (Roche Diagnostics, Basel, Switzerland) with SYBR® Green Master Mix (Takara Biotechnology). β-actin was used as an endogenous control. The primer sequences were as follows: GLS, 5′-TGGT GGCCTCAGGTGAAAAT-3′ (forward) and 5′-CCAA GCTAGGTAACAGACCCTGTTT-3′ (reverse); and β-actin, 5′-GATCATTGCTCCTCCTGAGC-3′ (forward) and 5′ACTCCTGCTTGCTGATCCAC-3′ (reverse). The relative expression of RNA was calculated with the ΔCT method.

Western blot analysis Total cell protein was extracted with a whole cell lysis assay kit (KeyGEN BioTECH, China) and was quantified using the BCA protein assay kit (Beyotime Biotechnology, China). Whole cell lysates were subjected to SDS-PAGE and transferred onto PVDF membranes. After being blocked with 5% skim milk (3% bovine serum albumin for p-p70s6k(Thr389) and p-mTOR(Ser-2448)), the membranes were incubated with primary antibodies overnight at 4 °C. HRP-conjugated anti-rabbit IgG (ZS Bio, China) and chemiluminescent HRP substrate (ECL, Millipore Corporation, Billerica, USA) were used to visualize proteins. The primary antibodies used were as follows: GLS (Abcam, Cambridge, UK); p70s6k, pp70s6k(Thr389) , mTOR, p-mTOR (Ser-2448) , ULK, and pULK1Ser757 (Cell Signaling Technology, USA); 4EBP1 (Bethyl Laboratories, Montgomery, TX); p-4EBP1Thr46 (Invitrogen, Camarillo, CA); and LC3B and p62 (SigmaAldrich, St. Louis, MO).

Cell apoptosis analysis The effect of GLS on the apoptosis of thyroid cancer cell lines was detected with an Annexin V-FITC Detection Kit (BioLegend, San Diego, CA) according to the manufacturer’s protocol. Cells were incubated with agents for indicated times and were treated with apoptosis inducers A (Apopisa) and B (Apobid) (1:2000; Beyotime, China) for 20 h before being collected along with the medium. After being resuspended in annexin V binding buffer, cells were double-stained with annexin V-FITC and propidium iodide (PI) to analyze apoptotic cells. The cells were detected with a FACSCalibur flow cytometer (Becton-Dickinson, San Diego, CA). FlowJo software (USA) was used to further analyze the flow cytometry results.

Statistical analysis The results are presented as the means ± standard deviation (SD). Statistical analysis, including Student’s t test, the MannWhitney test, and Wilcoxon’s signed-rank test, was performed using SPSS 20.0 software. Results with a P value of < 0.05 from at least three independent experiments were considered statistically significant.

Results PTC cell lines were glutamine-dependent To investigate the glutamine dependence of PTC cell lines, we analyzed the proliferation and viability of four different PTC cell lines (K1, IHH4, BCPAP, and TPC-1) and a noncancerous thyroid cell line (Nthy-ori 3-1) in growth media with and without glutamine. We found that glutamine deprivation exerted little effect on Nthy-ori 3-1 cells, while the proliferation and viability of all four PTC cell lines were significantly decreased in glutamine deprivation media (Figs. 1a and S1a, d), indicating that the PTC cell lines were glutaminedependent.

GLS was screened with RT2-PCR arrays Using commercially available RT2-PCR arrays, we conducted an RT2-PCR screen of 48 amino acid-associated genes with specific roles in the regulation of amino acid metabolism. Six pairs of fresh PTC tissues and matched non-tumor thyroid tissues and IHH4 and BCPAP cell lines were tested. mRNA from non-tumor thyroid tissues or the thyroid cell line Nthyori 3-1 was treated as a control. Table S1 showed all genes we tested with array, and we only selected genes that consistently altered in both IHH4 and BCPAP cells to be listed in Table 1.

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Fig. 1 Glutamine dependence of PTC cell lines and GLS expression in thyroid cell lines and tissues. a Effects of glutamine on the proliferation of PTC and noncancerous thyroid cells. Data are shown as the means ± SD (n = 5 per time point). The data represent three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. b The expression levels of GLS were determined by real-time PCR and Western blot in five thyroid cell lines. Data are shown as the means ± SD, n = 3. *P < 0.05. c Relative

expression levels of GLS in 90 pairs of human PTC tissues compared to m a t c h e d n o n - t u m o r t i s s u e s , P < 0 . 0 0 1 . d R e p r e s e n t at i v e immunohistochemistry images of GLS expression in non-tumor thyroid tissues (× 100 and × 400). e Representative immunohistochemistry images of GLS expression in PTC tissues (× 100 and × 400). f, g Immunohistochemical analyses of GLS expression in paired and nonpaired PTC tissue specimens, respectively

J Mol Med Table 1 Amino acid-associated genes identified by RT2-PCR arrays screening as differentially expressed in PTC tissues, IHH4, and BCPAP cells as compared to matched non-tumor thyroid tissues and Nthy-ori 3-1 cells Targeta

Description

PTC vs matched non-tumor tissues Fold change

P value

IHH4 vs NTb cell

BCPAP vs NTb cell

Fold change

Fold change

ALDH4A1

Aldehyde dehydrogenase 4 family member A1

− 1.8741

0.0276

− 2.3295

− 2.9485

ARG2 NOS2

Arginase 2 Nitric oxide synthase 2

− 1.3935 2.1810

0.0274 0.1600

2.7321 − 2.9691

3.7064 − 71.5064

GLS

Glutaminase

1.5105

0.0015

7.3615

2.2346

GLUL GLS2

Glutamate-ammonia ligase Glutaminase 2

− 1.5980 1.8056

0.0070 0.0010

5.6962 − 4.5631

10.7779 − 15.5625

PRODH2 GLUD1

Proline dehydrogenase 2 Glutamate dehydrogenase 1

− 1.1547 − 1.1454

0.6971 0.4050

1.1487 − 2.2974

1.7291 − 1.0353

AUH

AU RNA binding methylglutaconyl-CoA hydratase

− 1.1658

0.2147

− 2.0849

− 2.1435

The significance of the values has been indicated in italics a

Genes that consistently altered in both IHH4 and BCPAP cells were listed in Table 1

b

NT Nthy-ori 3-1, non-tumor thyroid cell line

GLS was the only gene that was consistently overexpressed in both PTC tissues and cell lines.

GLS was overexpressed in human PTC cell lines To confirm the results of the RT2-PCR arrays, we first explored the mRNA and protein expression levels of GLS in four PTC cell lines. The four PTC cell lines (K1, IHH4, BCPAP, and TPC-1) and the noncancerous thyroid cell line Nthy-ori 3-1 were tested. GLS was overexpressed in all four PTC cell lines compared to its expression in Nthy-ori 3-1 cells (Fig. 1b).

GLS was overexpressed in human PTC tissues We then investigated GLS expression in human PTC tissues. RT-PCR results suggested that GLS was highly expressed in 90 pairs of fresh human PTC tissues compared to the matched non-tumor adjacent thyroid tissues (P < 0.001, Fig. 1c). According to the results of the tissue microarray IHC of 54 pairs of PTC tissues, GLS-positive staining was found in 70.4% of PTC tissues and 3.7% of matched adjacent non-tumor thyroid tissues. GLS-positive Table 2

staining was also found in 60.4% of the 91 non-paired PTC tissues and 1.3% of the 76 non-tumor thyroid tissues. The staining scores of PTC tissues were significantly higher than those of the non-tumor thyroid tissues (P < 0.001, Fig. 1d–g and Table 2). Our results provided evidence that GLS was aberrantly overexpressed in human PTC at both the mRNA and protein levels.

Aberrant overexpression of GLS was correlated with clinicopathological characteristics Clinicopathological characteristics, including age, sex, maximal tumor size, multifocality, extrathyroidal extension (ETE), Hashimoto’s thyroiditis, lymph node metastasis, and TNM stage, were recorded and were analyzed for possible correlations with GLS expression. GLS expression levels were found to be positively associated with ETE (P = 0.005, Table 3). Notably, higher GLS expression levels were found in patients over 45 years old and patients with advanced TNM stages (P = 0.073 and 0.075, respectively). These results suggested that high GLS expression was correlated with invasive extension of PTC.

Immunoreactive staining of GLS in human thyroid tissues

Tissue type (paired)

Total (%)

GLSnegative (−/+)

PTC Non-tumor

54 (50.0) 16 (29.6) 54 (50.0) 52 (96.3)

P valuea

GLSpositive (++/+++)

IRS (mean ±S D)

38 (70.4) 2 (3.7)

8.0741 ± 4.6818 < 0.001 PTC 0.333 ± 1.1974 Non-tumor

Tissue type Total (non-paired) (%)

The significance of the values has been indicated in italics a

Comparisons between the IRS values in different groups. Data are presented as n (%)

GLSnegative (−/+)

91 (54.2) 36 (39.6) 76 (45.8) 75 (98.7)

P valuea

GLSpositive (++/+++)

IRS (mean ± SD)

55 (60.4) 1 (1.3)

7.0220 ± 4.9238 < 0.001 0.3158 ± 1.2459

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Inhibition of GLS suppressed glutaminolysis and modulated mitochondrial respiration GLS is a key enzyme in glutaminolysis; thus, alteration of its expression in PTC cells should affect glutamine metabolism. To test this hypothesis, BPTES and CB-839 (pharmacological inhibitors targeting both isoforms of GLS [27, 28]) and siRNA were used, and their effects were evaluated after 48 h. The inhibitory effects for GLS of pharmacological inhibitors were confirmed with Western blots (Figs. S2a, b), and the knockdown efficiency of siRNA was assessed with Western blots and real-time PCR (Fig. S2c, d). We found that BPTES, CB-839, and siRNA transfection all reduced the levels of intracellular α-KG and glutamate in PTC cells, including K1, IHH4, BCPAP, and TPC-1 cells (Fig. 2a, b), indicating the suppression of glutaminolysis after GLS inhibition. In addition, we evaluated mitochondrial function with an XFe 96 extracellular flux analyzer to further assess the effect of GLS inhibition in K1 and TPC1 cells. BPTES and CB-839 clearly decreased the basal Table 3

OCR and mitochondrial respiratory capacity in K1 and TPC-1 cells compared with DMSO-treated control cells (Fig. 2c, e). Similar results were obtained after GLS knockdown (Fig. 2d, f). Overall, these findings suggested that targeting GLS-mediated glutaminolysis impaired mitochondrial respiration and OCR in PTC cells.

Inhibition of GLS decreased the proliferation, migration, and invasion ability of PTC cells We used BPTES, CB-839, and siRNA transfection to study the function of GLS in four PTC cell lines (K1, IHH4, BCPAP, and TPC-1). Compared to DMSO, BPTES and CB839 evidently suppressed cell growth and viability in all four PTC cell lines (Figs. 3a and S1b, e). To further analyze the function of GLS in PTC, GLS was genetically impaired using siRNA. Knockdown of GLS resulted in decreased cell proliferation, viability, migration, and invasion abilities of all four PTC cell lines compared with negative controls and blank cells (Figs. 3b–j and S1c, f). These results indicated that

Correlations between GLS expression and clinicopathological variables in PTC tissues

Clinical features

Total (%)

GLS-negative (−/+, n = 52)

GLS-positive (++/+++, n = 93)

IRS (mean ± SD)

P valuea

Age < 45 ≧ 45

71 (49.0) 74 (51.0)

30 (57.7) 22 (42.3)

41 (44.1) 52 (55.9)

6.68 ± 5.03 8.12 ± 4.58

0.073

Sex Male Female Maximal tumor size (cm)

39 (26.9) 106 (73.1)

12 (23.1) 40 (76.9)

27 (29.0) 66 (71.0)

8.08 ± 4.76 7.17 ± 4.87

0.309

<2 2–4

64 (44.1) 68 (46.9)

21 (40.4) 25 (48.1)

43 (46.2) 43 (46.2)

7.56 ± 4.60 7.44 ± 4.99

0.835

>4 Multifocality No

13 (9.0)

6 (11.5)

7 (7.6)

6.54 ± 5.50

106 (73.1)

39 (75.0)

67 (72.0)

7.37 ± 4.99

Yes Extrathyroidal extension No Yes Hashimoto’s thyroiditis No Yes Lymph node metastasis No Yes TNM stages I/II III/IV

39 (26.9)

13 (25.0)

26 (28.0)

7.54 ± 4.49

102 (70.3) 43 (29.7)

42 (80.8) 10 (19.2)

60 (64.5) 33 (35.5)

6.70 ± 4.89 9.12 ± 4.34

0.005

116 (80.0) 29 (20.0)

38 (73.1) 14 (26.9)

78 (83.9) 15 (16.1)

7.76 ± 4.77 6.03 ± 5.00

0.107

57 (39.3) 88 (60.7)

15 (28.8) 37 (71.2)

42 (45.2) 51 (54.8)

8.14 ± 4.51 6.94 ± 5.02

0.201

94 (64.8) 51 (35.2)

36 (69.2) 16 (30.8)

58 (62.4) 35 (37.6)

6.93 ± 4.89 8.31 ± 4.68

0.075

The significance of the values has been indicated in italics a

Comparisons between the IRS values in different groups. Data are presented as n (%)

0.925

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R Fig. 2

Effects of BPTES-, CB-839-, and siRNA-induced GLS inhibition on glutaminolysis and mitochondrial respiration. a Alteration of α-KG levels upon GLS inhibition with BPTES or CB-839 treatment or GLS knockdown in K1, IHH4, BCPAP, and TPC-1 cells. The data represent three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. b Alteration of glutamate levels upon GLS inhibition with BPTES or CB839 treatment or GLS knockdown in K1, IHH4, BCPAP, and TPC-1 cells. The data represent three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. c, e. Normalized OCR per μg of cell protein in K1 and TPC-1 cells treated with BPTES, CB-839, or DMSO. Left graphs represent the real-time and absolute oxygen consumption of cells, and right graphs represent basal OCR and maximal OCR after calculation. The data represent at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. d, f Normalized OCR per μg of cell protein in siRNA-transfected K1 and TPC-1 cells. Left graphs represent the realtime and absolute oxygen consumption of cells, and right graphs represent basal OCR and maximal OCR after calculation. The data represent at least three independent experiments. *P < 0.05

GLS expression may play a vital role in the development and progression of PTC.

Inhibition of GLS deactivated the mTORC1 signaling pathway To determine the molecular mechanism of GLS in PTC, Western blotting was performed to examine the activation status of the mTORC1 signaling pathway. The protein complex mTORC1 is a key regulator of cell proliferation, protein translation, and apoptosis inhibition, as well as a glutamine sensor [29, 30]. P70s6k and 4EBP1 are major downstream targets and effectors of mTORC1 [20, 31]. ULK1 is a vital component that regulates autophagy initiation in response to mTORC1 inhibition, and mTORC1-mediated phosphorylation of ULK1deactivated ULK1 [32, 33]. We examined the phosphorylation of mTOR at Ser2448, p70s6k at Thr389, 4EBP1 at Thr46, and ULK1 at Ser757 to investigate the activity of the mTORC1 signaling pathway. As shown in Figs. 4 and S3, BPTES-, CB-839-, and siRNA-mediated attenuation of GLS expression remarkably inhibited the phosphorylation of mTOR at Ser2448, p70s6k at Thr389, 4EBP1 at Thr46, and ULK1 at Ser757 in the K1, IHH4, BCPAP, and TPC-1 cell lines. Our results demonstrated that the suppression of the mTORC1 pathway might contribute to the antitumor effect of GLS inhibition.

Inhibition of GLS-induced autophagy and apoptosis of PTC cells We then investigated whether the antitumor effect of attenuated GLS expression and mTORC1 signaling activated autophagy and/or apoptosis of PTC cell lines. After BPTES or CB839 treatment for 48 h, the expression levels of the autophagy markers LC3B-I/II and p62 were assessed in the K1, IHH4, BCPAP, and TPC-1 cell lines by Western blot. BPTES or CB839 treatment in PTC cells resulted in the prominent conversion of LC3B I to LC3B II and a reduction in p62 levels,

suggesting the induction of autophagy (Fig. 4a, b). Concomitantly, similar results were observed upon GLS knockdown in the four PTC cell lines (Figs. 4a and S3c). Moreover, flow cytometric analysis indicated that pharmacological inhibition or genetic knockdown of GLS induced apoptosis in all four PTC cell lines compared to negative control cells and blank cells (P < 0.05, Fig. 5). Collectively, the results above verified that GLS inhibition dramatically induced autophagy and apoptosis in PTC cell lines.

Discussion Although several studies have reported the oncogenesis mechanism of PTC, most of these studies concentrated on the abnormal activation of the MAPK and PI3K-AKT pathways and the aberrant methylation status of genes in these pathways. In this study, we focused on the role of glutamine in PTC and found that GLS, a key enzyme in glutaminolysis, was upregulated in PTC and played a crucial role in the development and advancement of PTC. These results identified a novel molecular player in the development of PTC. This study reports, for the first time, the glutamine dependence of four PTC cell lines and the antitumor effect of GLS in PTC. Based on the glutamine dependence of PTC cells, we used RT2-PCR arrays to screen for amino acid-associated genes in PTC tissues and cells. We found that GLS was aberrantly upregulated in PTC compared to non-tumor thyroid tissues and cells. Subsequently, the overexpression of GLS in PTC was verified through IHC, RT-PCR, and Western blot analyses in human PTC tissues and four PTC cell lines. The glutamine deprivation conditions in the present study were not strictly achieved, because normal serum contains tiny amount of glutamine. While it was still deprived (the majority of glutamine is in the media, not the serum), to achieve true deprivation of glutamine, using dialyzed serum in combination with glutamine-free media should be highly recommended. A recent study revealed the altered expression of glutamine metabolism-related proteins in various histopathological subtypes of thyroid cancer [25], but that study failed to include non-cancer thyroid tissues and to compare the differential expression levels with PTC. We verified the upregulation of both the mRNA and protein levels of GLS in PTC tissues and cells compared to those in non-tumor thyroid tissues and cells, while previous studies have verified the aberrant overexpression of GLS in other cancer types, including acute myeloid leukemia, colorectal cancers, and hepatocellular carcinoma [21, 34, 35]. Considering the above results, we speculated that the upregulation of GLS may not be confined to a certain type of tumor. Kim et al. found that GDH was overexpressed in 81.1% samples of PTC [25]. Given that GDH catalyzes the transition of glutamate to α-ketoglutarate in the process of glutaminolysis, this study provides additional evidence that

J Mol Med

Fig. 3 Effects of BPTES-, CB-839-, and siRNA-induced GLS inhibition on proliferation, migration, and invasion of PTC cell lines. a Cell proliferation was assessed daily for 3 days with the CCK-8 assay in K1, IHH4, BCPAP, and TPC-1 cells treated with BPTES, CB-839, or DMSO (control). Data are shown as the means ± SD (n = 5 per time point). The data represent three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. b Cell proliferation was assessed daily for 3 days with

the CCK-8 assay in siRNA-transfected K1, IHH4, BCPAP, and TPC-1 cells. Data are shown as the means ± SD (n = 5 per time point). The data represent three independent experiments. *P < 0.05. c–j Transwell assays were used to assay the involvement of GLS in K1, IHH4, BCPAP, and TPC-1 cell migration and invasion. Data are shown as the means ± SD, n = 3. *P < 0.05

glutamine-associated proteins are of vital importance in the development of PTC. Significantly elevated expression of GLS in PTC suggests that GLS could be a potential biomarker for identifying indeterminate thyroid nodules. However, further studies confirming the roles of GLS in other histological subtypes of thyroid cancers are warranted. We also demonstrated that GLS overexpression was associated with ETE (P = 0.005), which is thought to be a risk factor for recurrence and disease-specific death from PTC, even at minimal levels [36]. Unfortunately, we were unable to obtain long-term follow-up data from patients; thus, we could not examine the relationship between GLS expression and disease prognosis. Nevertheless, the higher expression of GLS in PTC with ETE suggested an incremental adverse effect of positive GLS expression on the prognosis and aggressiveness of PTC. In fact, GLS expression has been reported to be a poor prognostic factor in other types of malignancies,

such as hepatocellular carcinoma and breast cancer [35, 37]. Another interesting phenomenon worth researching is the correlation between GLS expression grade and uptake of the glutamine analogue 4-18F-(2S,4R) fluoroglutamine (18FFGln) on positron emission tomography (PET). Previous studies have revealed that gliomas exhibiting glutamine dependence showed higher 18F-FGln uptake than normal brains in glioma mouse models and that glutamine dependence delineated gliomas in patients with disease progression [38]. Furthermore, GLS activity in human breast cancer cell xenografts in mice could be monitored with 18F-FGln PET imaging [39], but we were unable to analyze this correlation due to a lack of preoperative 18F-FGln PET imaging. Finally, four PTC cell lines (K1, IHH4, BCPAP, and TPC1) were fully investigated to explore the function of GLS in PTC cells. GLS inhibition with two pharmacologic inhibitors and siRNA knockdown reduced the intracellular levels of

J Mol Med

Fig. 4 Effects of GLS inhibition on the mTORC1 signaling pathway and autophagy of PTC cell lines. a Representative Western blots of p-p70s6k (Thr389), p70s6k, p-mTOR (Ser-2448), mTOR, p62, and LC3B I/II in K1, IHH4, BCPAP, and TPC-1 cells treated with BPTES or DMSO. b Representative Western blots of p-p70s6k (Thr389), p70s6k, p-mTOR

(Ser-2448), mTOR, p62, and LC3B I/II in K1, IHH4, BCPAP, and TPC-1 cells treated with CB-839 or DMSO. c Representative Western blots of p-p70s6k (Thr389), p70s6k, p-mTOR (Ser-2448), mTOR, and p62 in siRNA-transfected K1, IHH4, BCPAP, and TPC-1 cells

glutamate and α-KG in PTC cells, indicating a decline in glutaminolysis. Blockade of GLS also decreased the basal OCR and mitochondrial respiratory capacity of PTC cells,

suggesting a reduction in the oxidative phosphorylation (OXPHOS) metabolic pathway. Targeting GLS could reduce the cell proliferation, viability, migration, and invasion

J Mol Med

Fig. 5 Effect of GLS inhibition on apoptosis of PTC cell lines. a–d Apoptosis was evaluated by flow cytometry in K1, IHH4, BCPAP, and TPC-1 cells treated with BPTES, CB-839, or DMSO. Data are shown as the means ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001. e–h

Apoptosis was evaluated by flow cytometry in siRNA-transfected K1, IHH4, BCPAP, and TPC-1 cells. Data are shown as the means ± SD, n = 3. *P < 0.05, **P < 0.01

abilities of K1, IHH4, BCPAP, and TPC-1 cells, as well as inducing apoptotic cell death and autophagy. The deactivation of the mTORC1 signaling pathway after GLS inhibition may be responsible for impaired cell growth. Although related studies have confirmed that glutaminolysis induced the activation of mTORC1 signaling via RRAG proteins, and subsequently inhibited autophagy [40], the regulatory role of glutaminolysis on mTORC1 signaling in PTC is still poorly studied. It is essential to illustrate if the mechanisms of the regulation could be universally applied to PTC, since the clinicobiological features of PTC are quite different from other types of cancer. Autophagy might serve a dual role in cancer depending on the tumor stage and context; in early stages, autophagy suppresses tumor development by clearing damaged cellular components, but in later stages, it promotes survival and the growth of established tumors by providing nutrients and energy [40, 41]. Fan et al. confirmed that PTC cells undergo autophagy to sustain the energy supply needed for cell survival and growth [42]. As a result, the induction of autophagy after inhibition of GLS may imply a pro-survival

response in PTC cells. The combined targeting of GLS and autophagy may have synergistic antitumor effects in PTC cells. Further investigations are warranted to determine the role of GLS expression in PTC. As indicated in previous studies, the glutamate generated from glutamine catalyzed by GLS is also a crucial precursor of a substantial amount of nonessential amino acids [43]. Enzymes involved in the production of these amino acids were found to be activated in cancer. For example, glutamate pyruvate transaminase (GPT), which catalyzes the transfer of glutamate’s amino group to produce alanine and facilitate the disposal of extra nitrogen, can be stimulated by the KRAS oncogene to promote cancer cell growth [44]. Hence, the function of GLS in PTC must be fairly complicated and needs to be further elucidated. In addition, the mechanism of the regulation of GLS expression specifically in PTC is worthy of further investigation. Considering our findings that aberrant overexpression of GLS is prevalent in PTC and that silencing of GLS can inhibit the viability of cancer cells via inhibition of the mTORC1 signaling pathway, targeting of GLS is an attractive approach

J Mol Med

for PTC treatment, especially for patients who respond poorly to traditional therapeutic means. This approach is supported by a growing number of studies, in which the compounds 968, BPTES, and CB-839 were shown to suppress cancer cell growth by inhibiting GLS [28, 45, 46]. Furthermore, clinical trials of CB-839 treatment of hematological and solid tumors have been initiated (http://clinicaltrials.gov, NCT02071888 and NCT02071862 et al.). Our results provide evidence to support a clinical trial of GLS inhibitors in PTC patients. In conclusion, this is the first report to demonstrate that PTC cells are sensitive to glutamine resulting from GLS upregulation. Blockade of GLS could suppress the process of glutaminolysis and modulate mitochondrial OXPHOS. GLS overexpression in PTC provides a growth advantage for PTC cells by simulating cell proliferation, viability, migration, and invasion. Inhibition of GLS could induce apoptosis and autophagy via the mTORC1 signaling pathway. Ultimately, GLS may serve as a potential biomarker and an attractive therapeutic target for PTC treatment, and GLS inhibitors may be promising therapeutic agents for PTC patients. Acknowledgments We thank the technician Chao Guan for his generous help with preparing paraffin-embedded tissue blocks. Funding information This work was supported by the Distinguished Young Scholars Growth Programs of Higher Education of Liaoning Province (grant number LJQ2015114), the Scientific Research Programs of Liaoning Educational Committee (grant number LK201625), the Natural Science Foundation of Liaoning Province (grant number 2014021039), the Endocrine Diseases Research Programs of Chinese Medical Association Clinical Medicine Scientific Research (grant number 13050810466), the Open Project Program of Key Laboratory for Tumor Precision Medicine of Shaanxi Province, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, People’s Republic of China (grant number KLTPM-SX2018-A2), and the Support Programs for Young Scientific and Technological Innovation Talents of Shenyang (grant number RC170058).

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Compliance with ethical standards The study was supported by the First Affiliated Hospital of China Medical University and was conducted in accordance with the Declaration of Helsinki. Tissue specimens were collected with patients’ informed consent according to institutional ethical guidelines approved by the Institute Research Ethics Committee. Conflict of interest The authors declare that they have no conflict of interest.

References 1.

2.

Costa R, Carneiro BA, Chandra S, Pai SG, Chae YK, Kaplan JB, Garrett HB, Agulnik M, Kopp PA, Giles FJ (2016) Spotlight on lenvatinib in the treatment of thyroid cancer: patient selection and perspectives. Drug Des Devel Ther 10:873–884 Kitahara CM, Sosa JA (2016) The changing incidence of thyroid cancer. Nat Rev Endocrinol 12:646–653

16. 17.

18.

19.

20.

21.

Lim H, Devesa SS, Sosa JA, Check D, Kitahara CM (2017) Trends in thyroid cancer incidence and mortality in the United States, 1974–2013. JAMA 317:1338–1348 Grogan RH, Kaplan SP, Cao H, Weiss RE, Degroot LJ, Simon CA, Embia OM, Angelos P, Kaplan EL, Schechter RB (2013) A study of recurrence and death from papillary thyroid cancer with 27 years of median follow-up. Surgery 154:1436–1446; discussion 1446– 1437 Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674 Jin L, Alesi GN, Kang S (2016) Glutaminolysis as a target for cancer therapy. Oncogene 35:3619–3625 Ngo H, Tortorella SM, Ververis K, Karagiannis TC (2015) The Warburg effect: molecular aspects and therapeutic possibilities. Mol Biol Rep 42:825–834 van Geldermalsen M, Wang Q, Nagarajah R, Marshall AD, Thoeng A, Gao D, Ritchie W, Feng Y, Bailey CG, Deng N, Harvey K, Beith JM, Selinger CI, O’Toole SA, Rasko JEJ, Holst J (2016) ASCT2/ SLC1A5 controls glutamine uptake and tumour growth in triplenegative basal-like breast cancer. Oncogene 35:3201–3208 Willems L, Jacque N, Jacquel A, Neveux N, Maciel TT, Lambert M, Schmitt A, Poulain L, Green AS, Uzunov M et al (2013) Inhibiting glutamine uptake represents an attractive new strategy for treating acute myeloid leukemia. Blood 122:3521–3532 Zhang J, Pavlova NN, Thompson CB (2017) Cancer cell metabolism: the essential role of the nonessential amino acid, glutamine. EMBO J 36:1302–1315 Bhutia YD, Babu E, Ramachandran S, Ganapathy V (2015) Amino acid transporters in cancer and their relevance to Bglutamine addiction^: novel targets for the design of a new class of anticancer drugs. Cancer Res 75:1782–1788 Katt WP, Cerione RA (2014) Glutaminase regulation in cancer cells: a druggable chain of events. Drug Discov Today 19:450–457 Mates JM, Segura JA, Martin-Rufian M, Campos-Sandoval JA, Alonso FJ, Marquez J (2013) Glutaminase isoenzymes as key regulators in metabolic and oxidative stress against cancer. Curr Mol Med 13:514–534 Ferreira AP, Cassago A, Goncalves Kde A, Dias MM, Adamoski D, Ascencao CF, Honorato RV, de Oliveira JF, Ferreira IM, Fornezari C et al (2013) Active glutaminase C self-assembles into a supratetrameric oligomer that can be disrupted by an allosteric inhibitor. J Biol Chem 288:28009–28020 Scalise M, Pochini L, Galluccio M, Indiveri C (2016) Glutamine transport. From energy supply to sensing and beyond. Blood 1857: 1147–1157 Pavlova NN, Thompson CB (2016) The emerging hallmarks of cancer metabolism. Cell Metab 23:27–47 Bhutia YD, Ganapathy V (2016) Glutamine transporters in mammalian cells and their functions in physiology and cancer. Biochim Biophys Acta 1863:2531–2539 Daye D, Wellen KE (2012) Metabolic reprogramming in cancer: unraveling the role of glutamine in tumorigenesis. Semin Cell Dev Biol 23:362–369 Duran RV, Oppliger W, Robitaille AM, Heiserich L, Skendaj R, Gottlieb E, Hall MN (2012) Glutaminolysis activates RagmTORC1 signaling. Mol Cell 47:349–358 Nicklin P, Bergman P, Zhang B, Triantafellow E, Wang H, Nyfeler B, Yang H, Hild M, Kung C, Wilson C, Myer VE, MacKeigan JP, Porter JA, Wang YK, Cantley LC, Finan PM, Murphy LO (2009) Bidirectional transport of amino acids regulates mTOR and autophagy. Cell 136:521–534 Matre P, Velez J, Jacamo R, Qi Y, Su X, Cai T, Chan SM, Lodi A, Sweeney SR, Ma H, Davis RE, Baran N, Haferlach T, Su X, Flores ER, Gonzalez D, Konoplev S, Samudio I, DiNardo C, Majeti R, Schimmer AD, Li W, Wang T, Tiziani S, Konopleva M (2016)

J Mol Med Inhibiting glutaminase in acute myeloid leukemia: metabolic dependency of selected AML subtypes. Oncotarget 7:79722–79735 22. Zhang J, Wang G, Mao Q, Li S, Xiong W, Lin Y, Ge J (2016) Glutamate dehydrogenase (GDH) regulates bioenergetics and redox homeostasis in human glioma. Oncotarget. https://doi.org/10. 18632/oncotarget.7657 23. White MA, Lin C, Rajapakshe K, Dong J, Shi Y, Tsouko E, Mukhopadhyay R, Jasso D, Dawood W, Coarfa C, Frigo DE (2017) Glutamine transporters are targets of multiple oncogenic signaling pathways in prostate cancer. Mol Cancer Res 15:1017– 1028 24. Elgogary A, Xu Q, Poore B, Alt J, Zimmermann SC, Zhao L, Fu J, Chen B, Xia S, Liu Y, Neisser M, Nguyen C, Lee R, Park JK, Reyes J, Hartung T, Rojas C, Rais R, Tsukamoto T, Semenza GL, Hanes J, Slusher BS, le A (2016) Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer. Proc Natl Acad Sci U S A 113:E5328–E5336 25. Kim HM, Lee YK, Koo JS (2016) Expression of glutamine metabolism-related proteins in thyroid cancer. Oncotarget 7: 53628–53641 26. Shi RL, Qu N, Luo TX, Xiang J, Liao T, Sun GH, Wang Y, Wang YL, Huang CP, Ji QH (2017) Programmed death-ligand 1 expression in papillary thyroid cancer and its correlation with clinicopathologic factors and recurrence. Thyroid 27:537–545 27. Robinson MM, McBryant SJ, Tsukamoto T, Rojas C, Ferraris DV, Hamilton SK, Hansen JC, Curthoys NP (2007) Novel mechanism of inhibition of rat kidney-type glutaminase by bis-2-(5phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES). Biochem J 406:407–414 28. Gross MI, Demo SD, Dennison JB, Chen L, Chernov-Rogan T, Goyal B, Janes JR, Laidig GJ, Lewis ER, Li J, MacKinnon AL, Parlati F, Rodriguez MLM, Shwonek PJ, Sjogren EB, Stanton TF, Wang T, Yang J, Zhao F, Bennett MK (2014) Antitumor activity of the glutaminase inhibitor CB-839 in triple-negative breast cancer. Mol Cancer Ther 13:890–901 29. Green DR, Galluzzi L, Kroemer G (2014) Cell biology. Metabolic control of cell death. Science 345:1250256 30. Duran RV, Hall MN (2012) Glutaminolysis feeds mTORC1. Cell Cycle 11:4107–4108 31. Faustino A, Couto JP, Populo H, Rocha AS, Pardal F, CameselleTeijeiro JM, Lopes JM, Sobrinho-Simoes M, Soares P (2012) mTOR pathway overactivation in BRAF mutated papillary thyroid carcinoma. J Clin Endocrinol Metab 97:E1139–E1149 32. Park JM, Seo M, Jung CH, Grunwald D, Stone M, Otto NM, Toso E, Ahn Y, Kyba M, Griffin TJ, Higgins LA, Kim DH (2018) ULK1 phosphorylates Ser30 of BECN1 in association with ATG14 to stimulate autophagy induction. Autophagy 1–14:584–597

33.

Kim J, Kundu M, Viollet B, Guan KL (2011) AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol 13:132–141 34. Lu WQ, Hu YY, Lin XP, Fan W (2017) Knockdown of PKM2 and GLS1 expression can significantly reverse oxaliplatin-resistance in colorectal cancer cells. Oncotarget 8:44171–44185 35. Yu D, Shi X, Meng G, Chen J, Yan C, Jiang Y, Wei J, Ding Y (2015) Kidney-type glutaminase (GLS1) is a biomarker for pathologic diagnosis and prognosis of hepatocellular carcinoma. Oncotarget 6: 7619–7631 36. Yin DT, Yu K, Lu RQ, Li X, Xu J, Lei M (2016) Prognostic impact of minimal extrathyroidal extension in papillary thyroid carcinoma. Medicine 95:e5794 37. Kim JY, Heo SH, Choi SK, Song IH, Park IA, Kim YA, Park HS, Park SY, Bang WS, Gong G, Lee HJ (2017) Glutaminase expression is a poor prognostic factor in node-positive triple-negative breast cancer patients with a high level of tumor-infiltrating lymphocytes. Virchows Arch 470:381–389 38. Venneti S, Dunphy MP, Zhang H, Pitter KL, Zanzonico P, Campos C, Carlin SD, La Rocca G, Lyashchenko S, Ploessl K et al (2015) Glutamine-based PET imaging facilitates enhanced metabolic evaluation of gliomas in vivo. Sci Transl Med 7:274ra217 39. Zhu L, Ploessl K, Zhou R, Mankoff D, Kung HF (2017) Metabolic imaging of glutamine in cancer. J Nucl Med 58:533–537 40. Villar VH, Merhi F, Djavaheri-Mergny M, Duran RV (2015) Glutaminolysis and autophagy in cancer. Autophagy 11:1198– 1208 41. Jiang X, Overholtzer M, Thompson CB (2015) Autophagy in cellular metabolism and cancer. J Clin Invest 125:47–54 42. Fan D, Liu SYW, van Hasselt CA, Vlantis AC, Ng EKW, Zhang H, Dong Y, Ng SK, Chu R, Chan ABW, du J, Wei W, Liu X, Liu Z, Xing M, Chen GG (2015) Estrogen receptor α induces prosurvival autophagy in papillary thyroid cancer via stimulating reactive oxygen species and extracellular signal regulated kinases. J Clin Endocrinol Metab 100:E561–E571 43. DeBerardinis RJ, Cheng T (2010) Q’s next: the diverse functions of glutamine in metabolism, cell biology and cancer. Oncogene 29: 313–324 44. Martinez-Outschoorn UE, Peiris-Pages M, Pestell RG, Sotgia F, Lisanti MP (2017) Cancer metabolism: a therapeutic perspective. Nat Rev Clin Oncol 14:11–31 45. Han T, Guo M, Zhang T, Gan M, Xie C, Wang JB (2017) A novel glutaminase inhibitor-968 inhibits the migration and proliferation of non-small cell lung cancer cells by targeting EGFR/ERK signaling pathway. Oncotarget 8:28063–28073 46. Xiang Y, Stine ZE, Xia J, Lu Y, O’Connor RS, Altman BJ, Hsieh AL, Gouw AM, Thomas AG, Gao P et al (2015) Targeted inhibition of tumor-specific glutaminase diminishes cell-autonomous tumorigenesis. J Clin Invest 125:2293–2306