Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Jun 15.
Published in final edited form as: Clin Cancer Res. 2015 Feb 27;21(12):2780–2791. doi: 10.1158/1078-0432.CCR-14-2792

Methionine Deprivation Induces a Targetable Vulnerability in Triple-negative Breast Cancer Cells by Enhancing TRAIL Receptor-2 Expression

Elena Strekalova 1, Dmitry Malin 1, David M Good 1,*, Vincent L Cryns 1
PMCID: PMC4470820  NIHMSID: NIHMS668702  PMID: 25724522

Abstract

Purpose

Many neoplasms are vulnerable to methionine deficiency by mechanisms that are poorly understood. Because gene profiling studies have revealed that methionine depletion increases TNF-related apoptosis-inducing ligand receptor-2 (TRAIL-R2) mRNA, we postulated that methionine stress sensitizes breast cancer cells to proapoptotic TRAIL-R2 agonists.

Experimental Design

Human triple (ER/PR/HER2)-negative breast carcinoma cell lines were cultured in control or methionine-free media. The effects of methionine depletion on TRAIL receptor expression and sensitivity to chemotherapy or a humanized agonistic TRAIL-R2 monoclonal antibody (lexatumumab) were determined. The melanoma-associated antigen MAGED2 was silenced to delineate its functional role in sensitizing TNBC cells to methionine stress. An orthotopic TNBC model was utilized to evaluate the effects of dietary methionine deficiency, lexatumumab or the combination.

Results

Methionine depletion sensitized TNBC cells to lexatumumab-induced caspase activation and apoptosis by increasing TRAIL-R2 mRNA and cell surface expression. MCF-10A cells transformed by oncogenic H-Ras, but not untransformed cells, and matrix-detached TNBC cells were highly sensitive to the combination of lexatumumab and methionine depletion. Proteomics analyses revealed that MAGED2, which has been reported to reduce TRAIL-R2 expression, was suppressed by methionine stress. Silencing MAGED2 recapitulated features of methionine deprivation, including enhanced mRNA and cell surface expression of TRAIL receptors and increased sensitivity to TRAIL receptor agonists. Dietary methionine deprivation enhanced the antitumor effects of lexatumumab in an orthotopic metastatic TNBC model.

Conclusion

Methionine depletion exposes a targetable defect in TNBC cells by increasing TRAIL-R2 expression. Our findings provide the foundation for a clinical trial combining dietary methionine restriction and TRAIL-R2 agonists.

Keywords: methionine depletion, breast cancer, TRAIL, nutrition, therapeutics

Introduction

Transformed cells are selectively vulnerable to specific amino acid deficiencies, including methionine, arginine, leucine, serine and others (1–4). Depletion of the essential amino acid methionine inhibits cell cycle progression and induces apoptosis in cancer cells in vitro and suppresses tumor growth in vivo in preclinical models of diverse tumor types (5–9). Strikingly, supplementation with homocysteine renders normal cells largely resistant to methionine depletion, while transformed cells remain sensitive to methionine deprivation in the presence of homocysteine (10, 11). In addition, administration of the methionine-degrading enzyme methioninase mimics many of the antitumor actions of methionine depletion in vitro and in vivo (1, 12, 13). Both methionine deprivation and methioninase have been reported to enhance the cytotoxicity of chemotherapy drugs in some but not all studies; these chemosensitizing effects have been attributed to methionine stress-induced cell cycle blockade (14–17). Methionine depletion reduces the free concentration of intracellular methionine despite normal rates of methionine synthesis from homocysteine in tumor cells (18, 19). Although methionine plays an integral role in many biochemical pathways including protein and polyamine synthesis and methylation of nucleic acids and proteins, the molecular mechanisms underlying the “methionine dependence” of many neoplasms remain poorly understood (20). Clearly, a more detailed understanding of the cellular response to methionine deprivation would greatly facilitate the development of more effective combination therapies that act synergistically with methionine stress.

Gene expression analyses have revealed that both tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its proapoptotic receptor TRAIL-R2 mRNA are upregulated in methionine-dependent CNS tumor cell lines in response to methionine depletion (21). Although the functional relevance of the observed increase in TRAIL/TRAIL-R2 mRNA was not explored, these findings suggest that methionine stress may sensitize cancer cells to proapoptotic TRAIL receptor agonists. TRAIL/Apo2L is a promising cancer therapy that preferentially induces apoptosis in transformed cells by binding to its proapoptotic death receptors, TRAIL-R1/DR4 and TRAIL-R2/DR5, and activating procapases-8/-10 by a FADD-dependent mechanism in the extrinsic apoptotic pathway (22). Moreover, TRAIL and agonistic monoclonal antibodies (mAbs) targeting TRAIL-R1 or TRAIL-R2 inhibit primary tumor growth and metastatic tumor burden in preclinical models of diverse tumor types including breast cancer (23–28). We have recently reported that a human mAb targeting TRAIL-R2 (lexatumumab) is more effective than an agonistic TRAIL-R1 mAb (mapatumumab) in inducing apoptosis and suppressing lung metastases in an orthotopic model of clinically aggressive triple (ER/PR/HER2)-negative breast cancer (28). Recently, recombinant TRAIL (dulanermin) and agonistic mAbs targeting TRAIL-R1 or TRAIL-R2 have been evaluated in clinical trials in patients with advanced malignancies (29–34). Although these early stage clinical trials have demonstrated the safety and tolerability of TRAIL receptor agonists, they have been largely disappointing from a therapeutic standpoint (35). We postulated that methionine deprivation would enhance the sensitivity of triple-negative breast cancer (TNBC) cells to TRAIL-R2 targeted therapies such as lexatumumab and augment its antitumor activity in vivo.

Here we report that methionine depletion robustly sensitizes a panel of human TNBC cells to lexatumumab-induced caspase activation and apoptosis by increasing TRAIL-R2 mRNA and cell surface expression. Strikingly, MCF-10A cells transformed by oncogenic H-Ras, but not untransformed cells, were highly sensitive to the combination of lexatumumab and methionine depletion. Proteomics analyses revealed that the melanoma-associated antigen MAGED2, which has been reported to inhibit TRAIL-induced apoptosis by reducing TRAIL-R2 expression (36), was suppressed by methionine depletion. Silencing MAGED2 recapitulated features of methionine deprivation, including enhanced TRAIL receptor expression and increased sensitivity to TRAIL receptor agonists. In addition, dietary methionine deprivation enhanced the antitumor effects of lexatumumab in an orthotopic model of metastatic TNBC. Collectively, our findings indicate that methionine stress selectively induces a targetable vulnerability in transformed cells by increasing TRAIL-R2 expression.

Material and Methods

Cell culture and reagents

Human MDA-MB-231 TNBC cells stably expressing mCherry fluorescent protein and GILM2 TNBC cells were cultured as described previously (28). MDA-MB-468 TNBC cells stably expressing mCherry fluoresecent protein were generated by lentiviral transduction as described (37) and were maintained in DMEM supplemented with 10% FBS and 100 IU/mL penicillin/streptomycin (Invitrogen). Human MCF-10A breast epithelial cells stably expressing H-RasV12 or empty vector were cultured as described previously (38). For methionine deprivation, complete medium was formulated by supplementing RPMI 1640 with additional nutrients to closely match the original media for each cell line. Methionine-free medium (0% Met) was formulated in the same way as complete medium, without addition of 15 mg/L L-methionine. Lexatumumab was generously supplied by Dr. Robin Humphreys (Human Genome Sciences). Doxorubicin, Vinorelbine, 5-Fluorouracil, Gemcitabine and Docetaxel were purchased from Sigma-Aldrich.

Immunoblotting

Proteins were immunoblotted from whole-cell lysates as described (38). Primary Abs against MAGED2, β-actin (Sigma-Aldrich), PARP (BD Biosciences) and caspase-3 (Cell Signaling Technology) were used.

Cell viability assay

An MTS assay (Promega) was used to measure cell viability as described (28). Cells were plated in 96-well plates (2.5 × 103 cells/well) overnight. The next day, cells were washed with PBS, and complete or 0% Met medium was added. After 72 hours, MDA-MB-468 and GILM2 cells were treated with doxorubicin (3.4 μM), lexatumumab (4 μg/ml), gemcitabine (4 μM), vinorelbine (4 μM), docetaxel (4 nM) or 5-Fluorouracil (4 μg/ml), while MDA-MB-231 cells were treated with doxorubicin (2 μM), lexatumumab (2 μg/ml), gemcitabine (2 μM), vinorelbine (2 μM), docetaxel (2 nM) or 5-Fluorouracil (2 μg/ml). Cell viability was assessed after 48 hours of drug treatment. Cell viability was expressed as the percentage of viable cells compared to control vehicle-treated cells in complete medium.

Crystal violet cell survival assay

Cells were seeded on 6-well plates (3 × 105 cells/well). The next day, cells were washed with PBS, and complete or 0% Met medium was added. After 24 hours, cells were treated with vehicle, doxorubicin (3.4 μM) or lexatumumab (4 μg/ml) for MDA-MB-468 and GILM2 cells or with vehicle, doxorubicin (2 μM) or lexatumumab (2 μg/ml) for MDA-MB-231 cells for 48 hours. MCF-10A cells were treated with vehicle, doxorubicin (3.4 μM) or lexatumumab (2 μg/ml) for 24 hours. Surviving cells were stained with crystal violet as described (26).

Flow cytometry

For the Annexin V apoptosis assay, cells were cultured in complete or 0% Met medium for 48 hours, treated with vehicle, doxorubicin (3.4 μM) or lexatumumab (2 μg/ml) for 6 hours, and then analyzed by flow cytometry using the Annexin-PE Apoptosis Detection Kit (BD Bioscience). To measure cell surface expression of TRAIL receptors, cells were washed once with PBS and incubated with TRAIL-R1, TRAIL-R2 or control IgG1 mAb conjugated with fluorescein isothiocyanate (Abcam) in PBS containing 2.5% BSA for 45 minutes at 4°C in the dark. Cells were then washed 3 times with PBS containing 2.5% BSA, resuspended in PBS containing 4′,6-diamidino-2-phenylindole ( DAPI, 1 μg/mL), and analyzed by flow cytometry.

Anoikis assay

mCherry-labeled MDA-MB-231 cells were grown in complete growth medium or 0% Met medium containing 1% methylcellulose on Corning Costar Ultra-Low attachment plates (Fisher Scientific) at a density of 1.0 × 104 cells/well (96-well plates) for 48 hours prior to drug treatment (MTS assay) or 2.0 × 105 cells/well (6-well plates) for 72 hours (flow cytometry).

MAGED2 siRNA experiments

siRNAs targeting the sequences GGGAUACAUCAUUCACUCU (MAGED2-1) or GGGCAAAUGAUUUGGUGAA (MAGED2-2) of human MAGED2 and non-silencing control siRNA were purchased from Sigma-Aldrich. Cells were transfected with siRNAs using Lipofectamine RNAiMAX Reagent (Invitrogen).

Real-time PCR

cDNA was synthesized from total RNA using iScript™ cDNA Synthesis Kit (Bio-Rad). Primers for MAGE-D2 (forward 5-TCAGCACCTTAGAGCCCACT-3, reverse 5-CAGATGACAGCCTCACTGGA-3), TRAIL-R1 (5-CAGAGGGATGGTCAAGGTCAAGG-3, reverse 5-CCACAACCTGAGCCGATGC-3), TRAIL-R2 (forward 5-CGCTGCACCAGGTGTGATT-3, reverse 5-GTGCCTTCTTCGCACTGACA-3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5-GAAGGTGAAGGTCGGAGTC-3, reverse 5-GAAGATGGTGATGGGATTTC-3) were purchased from Integrated DNA Technologies. PCR amplification was performed using iQ™ SYBR Green supermix (Bio-Rad) and a CFX96 Real Time PCR sequence detection system (Bio-Rad). Experiments were performed twice, each in triplicate. A comparative Ct method was used to compare the RNA expression in samples to that of the control in each experiment.

Orthotopic model of metastatic TNBC

All animal experiments were approved by the IACUC at the University of Wisconsin-Madison. MDA-MB-468-mCherry TNBC cells (2 × 106) in 100% Matrigel (BD Bioscience) were injected into the ducts of the 4th mammary glands of 4- to 5-week-old female NOD scid IL2 receptor γ chain knockout (NSG) mice (Jackson Laboratory) as described (37). Mice were randomized into four treatment groups (10 mice per group): (1) a control 15% protein diet (Teklad TD.01084) plus vehicle (PBS i.p. twice weekly, 6 doses), (2) control diet plus lexatumumab (10 mg/kg i.p. twice weekly, 6 doses), (3) an isocaloric 15% protein methionine-free (0% methionine, Teklad TD.140119) diet plus vehicle, or (4) a methionine-free diet plus lexatumumab (10 mg/kg i.p. twice weekly, 6 doses). The composition of each diet is listed (Supplementary Table S1). Mice were placed on their respective diets 8 weeks after tumor cell inoculation, and treatment (vehicle or lexatumumab) was initiated 0.5 weeks later. The diets were continued throughout the treatment period. Mammary tumors were measured with calipers and tumor volume was calculated as described (39). mCherry-fluorescent metastases were visualized in isolated whole lungs using a Leica MZ10F fluorescent stereomicroscope and tumor burden was scored using NIH ImageJ analysis as described (28). For quantitating the number of lung macrometastases, only metastases (≥ 0.1 mm) were scored.

Statistics

ANOVAs with Bonferroni posttests were done using Prism 4 GraphPad Software to assess statistical significance.

Results

Methionine depletion sensitizes TNBC cells to lexatumumab

To determine whether methionine depletion enhances the cytotoxicity of lexatumumab and diverse chemotherapy agents, three human TNBC cell lines (MDA-MB-231-mCherry, GILM2 and MDA-MB-468) were grown in control or methionine-free media for 72 hours and then treated with vehicle, lexatumumab, doxorubicin, gemcitabine, vinorelbine, docetaxel or 5-fluorouracil. Methionine deprivation alone was moderately cytotoxic against two of the three TNBC cell lines as determined by an MTS cell viability assay (Fig. 1A) and inhibited cell proliferation in all three TNBC cell lines as determined by a reduction in Ki67-positive cells (Supplementary Fig. S1). Of all the cancer therapeutics evaluated, only the combination of methionine depletion and lexatumumab resulted in enhanced cytotoxicity in all three TNBC cells compared to treatment in control media. To confirm these findings using a second assay, TNBC cells were grown in control or methionine-free media for 72 hours, then treated with vehicle, doxorubicin or lexatumumab for 48 hours, and viable cells scored by crystal violet staining. Under these conditions, methionine depletion dramatically sensitized TNBC cells to lexatumumab, but had no or little effect on the cytotoxicity of doxorubicin (Fig. 1B). Pretreatment of TNBC cells with methioninase, an enzyme that degrades methionine, also sensitized TNBC cells to lexatumumab (Supplementary Fig. S2A). In addition, MCF-10A breast epithelial cells transformed by oncogenic H-RasV12 were more sensitive to methionine depletion alone or the combination of methionine depletion and lexatumumab than untransformed MCF-10A cells expressing empty vector (Fig. 1C). Collectively, these results indicate that methionine depletion robustly sensitizes TNBC cells to the proapoptotic death receptor agonist lexatumumab and point to the potential tumor-selectivity of this combination.

Figure 1. Methionine deprivation sensitizes TNBC cells to lexatumumab.

Figure 1

Open in a new tab

A, MTS cell viability assay of TNBC cells cultured in normal growth media (control) or methionine-free media (0% Met) and treated for 48 hours with vehicle, doxorubicin (3.4 μM), lexatumumab (4 μg/ml), gemcitabine (4 μM), vinorelbine (4 μM), docetaxel (4 nM) or 5-Fluorouracil (4 μg/ml) for MDA-MB-468 and GILM2 cells or doxorubicin (2 μM), lexatumumab (2 μg/ml), gemcitabine (2 μM), vinorelbine (2 μM), docetaxel (2 nM) or 5-Fluorouracil (2 μg/ml) for MDA-MB-231 cells (mean ± SEM, n = 3). B, Crystal violet cell survival assay of TNBC cells cultured in control or 0% Met media and treated for 48 hours with vehicle, doxorubicin (3.4 μM) or lexatumumab (4 μg/ml) for MDA-MB-468 and GILM2 cells or doxorubicin (2 μM) or lexatumumab (2 μg/ml) for MDA-MB-231 cells. Left panel: representative images. Right panel: quantification performed by counting cells in 3 fields of each well (mean ± SEM, n = 3). C, Crystal violet cell survival assay of MCF-10A breast epithelial cells stably expressing oncogenic H-RasV12 or empty vector grown in 0% Met media and treated with vehicle, doxorubicin (3.4 μM) or lexatumumab (2 μg/ml) for 24 hours. Left panel: representative images. Right panel: quantification performed by counting cells in 3 fields of each well (mean ± SEM, n = 3). In all panels, *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Methionine deprivation augments lexatumumab-induced caspase activation and apoptosis in TNBC cells

To determine whether methionine deprivation enhances caspase activation by lexatumumab, TNBC cells were grown in control or methionine-free media, treated with vehicle, doxorubicin or lexatumumab, and then analyzed by immunoblotting. Methionine deprivation resulted in more robust proteolysis of the caspase substrate PARP as detected by decreased full-length PARP and/or increased cleaved product compared to TNBCs grown in control media (Fig. 2A). In addition, methionine deprivation augmented lexatumumab-induced proteolytic processing of procaspase-3 as detected by diminished procaspase-3 expression. In contrast, methionine restriction had little or no effect on doxorubicin-induced PARP cleavage or procaspase-3 processing under the conditions tested. Moreover, pretreatment of TNBC cells with methioninase augmented lexatumumab-induced PARP cleavage (Supplementary Fig. S2B). These findings indicate that methionine restriction enhances caspase activation by lexatumumab.

Figure 2. Methionine deprivation augments lexatumumab-induced caspase activation and apoptosis in TNBC cells.

Figure 2

Open in a new tab

A, TNBC cells were grown in control or 0% Met media for 48 hours and then treated for 24 hours with vehicle, doxorubicin (3.4 μM) or lexatumumab (2 μg/ml) for MDA-MB-468 and GILM2 cells or doxorubicin (2 μM) and lexatumumab (1 μg/ml) for MDA-MB-231 cells. Procaspase-3, PARP, and cleaved PARP were detected by immunoblotting. B, TNBC cells were cultured in control or 0% Met media and treated with vehicle, doxorubicin (3.4 μM) or lexatumumab (2 μg/ml) for 6 hours. Apoptosis was measured by Annexin V labeling using flow cytometry. C, MCF-10A breast epithelial cells stably expressing oncogenic H-RasV12 or empty vector were grown in control or 0% Met media and were treated with vehicle, doxorubicin (3.4 μM) or lexatumumab (2 μg/ml) for 6 hours. Apoptosis was quantitated by Annexin V labeling.

To quantitate the effect of methionine restriction on apoptosis, TNBC cells were grown in methionine-free media for 48 hours, treated with vehicle, doxorubicin or lexatumumab for 6 hours, and Annexin V-positive cells were scored by flow cytometry. Consistent with our MTS and crystal violet data, methionine depletion profoundly sensitized all three TNBC cell lines to lexatumumab-induced apoptosis (Fig. 2B). In contrast, methionine deprivation had little effect on doxorubicin-induced cell death. Furthermore, transformed MCF-10A-H-RasV12 cells were more sensitive to methionine depletion and the combination of methionine depletion and lexatumumab than untransformed MCF-10A-Vector cells, confirming the preferential proapoptotic activity of this treatment against cancer cells (Fig. 2C). However, methionine deprivation had little effect on doxorubicin-induced cell death in either transformed or untransformed MCF-10A cells. Taken together, these findings indicate that methionine depletion potently augments caspase activation and apoptosis by lexatumumab and underscores the enhanced sensitivity of transformed cells to this combination.

Methionine deprivation preferentially increases the cell surface expression of TRAIL-R2 in TNBC cells and sensitizes matrix-detached cells to lexatumumab

To delineate the mechanisms by which methionine depletion enhances the sensitivity of TNBC cells to lexatumumab, we first examined its effects on the expression of TRAIL-R1 and TRAIL-R2. Methionine deprivation for 72 hours resulted in robust induction of TRAIL-R2 and TRAIL-R1 mRNA levels in all three TNBC cell lines as determined by real-time PCR (Fig. 3A). Similarly, treatment of TNBC cells with methioninase for 48 hours increased TRAIL-R1 and TRAIL-R2 mRNA levels (Supplementary Fig. S2C). Moreover, methionine depletion increased the cell surface expression of TRAIL-R2 in all TNBC cell lines as determined by flow cytometry but had modest or no effect on TRAIL-R1 cell surface expression (Fig. 3B). Intriguingly, methionine deprivation increased the cell surface expression of both TRAIL-R1 and TRAIL-R2 in transformed MCF-10A-H-RasV12 cells but not in untransformed MCF-10A-Vector cells (Fig. 3C). These results suggest that methionine restriction sensitizes TNBC cells to lexatumumab by increasing the cell surface expression of TRAIL-R2.

Figure 3. Methionine deprivation preferentially increases the cell surface expression of TRAIL-R2 in TNBC cells and sensitizes matrix-detached cells to lexatumumab.

Figure 3

Open in a new tab

A, TNBC cells were cultured in control or 0% Met media for 72 hours followed by isolation of total RNA. TRAIL-R2 (left) and TRAIL-R1 (right) mRNA levels were measured by Real-time PCR and normalized to expression in TNBC cells grown in control media. B and C, Cell surface expression of TRAIL-R1 and TRAIL-R2 in TNBC cells (B) or MCF-10A breast epithelial cells stably expressing oncogenic H-RasV12 or empty vector (C). Cells were grown in control or 0% Met media for 72 hours, incubated with control IgG, TRAIL-R1 or TRAIL-R2 mAb, and analyzed by flow cytometry. Grey bar: negative control cells incubated with IgG. Black line: cells cultured in control media and incubated with TRAIL-R1 (bottom panels) or TRAIL-R2 (top panels) Ab. Red line: cells grown in 0% Met media and incubated with TRAIL-R1 (bottom panels) or TRAIL-R2 (top panels) Ab. D, Cell surface expression of TRAIL-R1 and TRAIL-R2 in mCherry-labeled MDA-MB-231 cells grown in suspension in control or 0% Met media. Cells were incubated with control IgG, TRAIL-R1 or TRAIL-R2 mAb and analyzed by flow cytometry. Grey bar: negative control adherent cells incubated with IgG. Black line: cells cultured in suspension in control media and incubated with TRAIL-R1 (bottom panel) or TRAIL-R2 (top panel) Ab. Red line: cells grown in suspension in 0% Met media and incubated with TRAIL-R1 (bottom panel) or TRAIL-R2 (top panel) Ab. E, MTS cell viability assay of mCherry-labeled MDA-MB-231 cells grown in suspension in control or methionine-free media and treated with vehicle of lexatumumab. Cells were grown in complete growth medium (control) or methionine-free media (0% Met) containing 1% methylcellulose on Ultra-Low attachment plates for 48 hours and then treated with vehicle or lexatumumab (1 μg/ml) for 24 hours (mean ± SEM, n = 3). **, P < 0.01

Because resistance to extracellular matrix detachment-induced apoptosis (“anoikis”) is a hallmark of metastatic carcinoma cells (40), we examined whether methionine deprivation enhanced cell surface expression of TRAIL-R2 and sensitized matrix-detached cells to lexatumumab. Similar to the results obtained in adherent cells, methionine depletion increased cell surface expression of TRAIL-R2 to a greater extent than TRAIL-R1 in mCherry-labeled MDA-MB-231 cells grown in suspension on Ultra-low attachment plates (Fig. 3D). Moreover, methionine depletion sensitized matrix-detached mCherry-labeled-MDA-MB-231 TNBC cells to lexatumumab as determined by MTS assay (Fig. 3E). Collectively, these results indicate that methionine depletion sensitizes matrix-detached TNBC cells to lexatumumab, supporting an antimetastatic activity for this novel combination.

Methionine deprivation reduces MAGED2 protein levels but does not affect MAGED2 mRNA levels

To identify proteins that might mediate the effects of methionine depletion on TRAIL receptor expression, we performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) of MDA-MB-231-mCherry and GILM2 TNBC cells grown in methionine-free or control media for 72 hours. Proteomics analysis revealed a number of proteins that were differentially expressed in TNBC cells in response to methionine depletion (Supplementary Table S2). One particularly intriguing candidate was MAGED2/MAGD2, which was downregulated by methionine depletion in both TNBC cell lines and has been previously reported to inhibit TRAIL-induced apoptosis by reducing TRAIL-R2 expression (36). Immunoblotting confirmed that MAGED2 protein levels were decreased in MDA-MB-231-mCherry, GILM2 and MDA-MB-468 TNBC cells grown in methionine-free media compared to cells cultured in control media (Fig. 4A). In contrast, methionine depletion did not affect MAGED2 mRNA levels (Fig. 4B), indicating that methionine deprivation regulates MAGED2 via a post-transcriptional mechanism.

Figure 4. Methionine deprivation reduces MAGED2 protein levels but does not affect MAGED2 mRNA levels.

Figure 4

Open in a new tab

A, Immunoblot of MAGED2 protein expression in TNBC cells grown in control (Met +) or 0% Met (−Met) media for 72 hours. B, Real-time PCR analysis of MAGED2 mRNA levels in TNBC cells grown in control or 0% Met media for 72 hours. MAGED2 mRNA levels were normalized to expression in TNBC cells cultured in control media.

Silencing MAGED2 increases cell surface expression of TRAIL receptors and sensitizes TNBC cells to TRAIL receptor agonists

To investigate the functional role of MAGED2 downregulation in the lexatumumab-sensitizing effects of methionine depletion, we transiently silenced MAGED2 in MDA-MB-231-mCherry cells using two different siRNAs (si-MAGED2-1 and si-MAGED2-2) targeting MAGED2. Both MAGED2 siRNAs robustly reduced MAGED2 mRNA and protein levels compared to a control scrambled siRNA (Fig. 5A). Silencing MAGED2 increased TRAIL-R1 and TRAIL-R2 mRNA levels (Fig. 5B) and cell surface expression of both death receptors (Fig. 5C). Consistent with these effects, silencing MAGED2 sensitized MDA-MB-231 TNBC cells to lexatumumab and the TRAIL-R1 agonist mapatumumab as determined by crystal violet staining (Fig. 5D) and MTS assay (Fig. 5E). Collectively, these results indicate that silencing MAGED2 mimics many of the effects of methionine depletion, including enhanced cell surface expression of TRAIL receptor and increased sensitivity to TRAIL receptor agonists.

Figure 5. Silencing MAGED2 increases cell surface expression of TRAIL receptors and sensitizes TNBC cells to TRAIL receptor agonists.

Figure 5

Open in a new tab

A, MDA-MB-231 cells were transfected with a scrambled control siRNA or one of two different siRNAs targeting MAGED2 (MAGED2-1 and MAGED2-2). Left panel: Real-time PCR analysis of MAGED2 mRNA levels 24 hours after siRNA transfection. MAGED2 mRNA levels were normalized to control siRNA-transfected cells. Right panel: Immunoblot analysis of MAGED2 48 hours after siRNA transfection. B, Real-time PCR analysis of TRAIL-R1 and TRAIL-R2 mRNA levels in MDA-MB-231 cells transfected with control or MAGED2 siRNAs. C, Cell surface expression of TRAIL-R1 and TRAIL-R2 in MDA-MB-231 cells transfected with control or MAGED2 siRNAs. Grey bar: negative control. Black line: control siRNA. Red line: MAGED2 siRNA. D, Crystal violet cell survival assay of MDA-MB-231 cells transfected with control or MAGED2 siRNA and treated with vehicle, lexatumumab (0.1 μg/ml) or mapatumumab (0.1 μg/ml) for 72 hours. Left panel: representative images. Right panel: quantification performed by counting cells in 3 fields of each well (mean ± SEM, n = 3). E, MTS cell viability assay of MDA-MB-231 cells transfected with control or MAGED2 siRNA and treated with vehicle, lexatumumab (1 μg/ml) or mapatumumab (1 μg/ml) for 24 hours (mean ± SEM, n=3). In (D) and (E), *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Dietary methionine deprivation enhances the antitumor effects of lexatumumab in an orthotopic model of metastatic TNBC

To examine the antitumor activity of methionine deprivation in combination with lexatumumab in vivo, female NSG mice with established MDA-MB-468-mCherry mammary tumors were fed a control or methionine-free diet (Supplementary Table S1) and treated with vehicle or lexatumumab (10 mg/kg twice weekly) for 3 weeks. All three interventions (methionine-free diet, lexatumumab alone, and methionine-free diet plus lexatumumab) inhibited tumor growth compared to vehicle-treated mice on a control diet, although the combination of methionine-free diet plus lexatumumab was more effective than diet alone or lexatumumab alone at the completion of the study (Fig. 6A). Both the methionine-free diet and the methionine-free diet plus lexatumumab resulted in modest weight loss (Fig. 6B). To evaluate the impact of treatment on lung metastases in vivo, mice were euthanized 13 weeks after intraductal tumor inoculation, and mCherry-fluorescent metastatic lesions in the lungs were identified at autopsy. Notably, only mice receiving the methionine-free diet in combination with lexatumumab had a significant reduction in tumor burden as defined as the percentage surface area occupied by lung metastases or number of macrometastases ≥ 0.1 mm compared to vehicle-treated mice on control diets (Fig. 6C). TRAIL-R2 mRNA levels were increased in mammary tumors from mice treated with the methionine-free diet compared to mice on a control diet (Fig. 6D), while MAGED2 protein levels were reduced in mammary tumors from mice on the methionine-free diet (Fig. 6E). These findings indicate that dietary methionine deprivation inhibits mammary tumor expression of MAGED2 and enhances TRAIL-R2 expression and the antitumor effects of lexatumumab in an orthotopic model of metastatic TNBC.

Figure 6. Dietary methionine deprivation enhances the antitumor effects of lexatumumab in an orthotopic model of metastatic triple-negative breast cancer.

Figure 6

Open in a new tab

Female NSG mice with orthotopic MDA-MB-468-mCherry mammary tumors were randomized into four groups (10 mice per group): control diet plus vehicle, control diet plus lexatumumab (10 mg/kg twice weekly for 6 doses, indicated by arrows), methionine-free diet plus vehicle, or methionine-free diet plus lexatumumab (10 mg/kg twice weekly for 6 doses). Mice were started on their respective diets 8 weeks after tumor cell inoculation, and the diets were continued throughout the treatment period. A, percentage original mammary tumor volume (at 8 weeks) in each treatment group (mean ± SEM, n = 10 mice per group). B, Body weight of mice. C, Representative whole lung images by fluorescence microscopy. The percentage of the surface area occupied by lung metastases and the number of fluorescent macrometastases per lung (mean ± SEM, n = 10 mice per group). D, Real-time PCR analysis of TRAIL-R2 mRNA levels in mammary tumors isolated from mice receiving control diet or methionine-free diet. mRNA levels were normalized to expression in mice receiving the control diet. E, Immunoblot of MAGED2 expression in mammary tumors from mice receiving control diet or methionine-free diet. In (A) through (C), *, P < 0.05, **, P < 0.01, ***, P < 0.001 versus vehicle-treated mice.

Discussion

We have demonstrated that methionine deprivation unmasks a targetable vulnerability of transformed cells by augmenting TRAIL-R2 mRNA levels and cell surface expression, resulting in enhanced sensitivity to proapoptotic TRAIL-R2 agonists such as lexatumumab. Similar results were obtained when we used methioninase to degrade methionine, a therapeutic strategy that has shown promise in preclinical studies (1, 12, 13, 15). Strikingly, the effects of methionine depletion on increasing cell surface expression of TRAIL-R2 and enhancing sensitivity to lexatumumab are largely confined to transformed cells, as untransformed MCF-10A-Vector cells were resistant to these effects. These findings are consistent with the reported tumor-selectivity of methionine deprivation and TRAIL receptor agonists when used individually (10, 11, 22, 24, 26, 41). Methionine depletion also sensitized matrix-detached TNBC cells to lexatumumab, supporting an antimetastatic activity for this combination. In addition, we have shown that a brief exposure to a methionine-free diet is well tolerated by mice and enhances the antitumor activity of lexatumumab against mammary tumors and lung metastases in vivo in an orthotopic MDA-MB-468 TNBC model that is partially resistant to lexatumumab alone. Intriguingly, this combination therapy suppressed lung metastases more robustly than it inhibited mammary tumor growth, consistent with the reported function of TRAIL as a metastasis suppressor and our prior preclinical observations with lexatumumab monotherapy in a different orthotopic model of metastatic TNBC (MDA-MB-231) that was sensitive to this agent (28, 42, 43). Our current findings point to methionine deprivation as a novel nutritional intervention to augment the antitumor efficacy of proapoptotic TRAIL receptor agonists, which have been largely disappointing in clinical trials (29–34). Moreover, phase I and phase II trials of a methionine-free diet alone or in combination with chemotherapy in patients with metastatic or recurrent solid tumors have confirmed the safety and tolerability of intermittent or continuous short-term methionine deprivation (44–46), suggesting that our findings could be readily translated into a clinical trial. Collectively, our results indicate that methionine depletion induces a selective vulnerability to lexatumumab by increasing expression of its molecular target and strongly support combining these interventions to enhance the therapeutic impact of TRAIL-R2 agonists. In support of this idea, we have recently demonstrated that metastatic TNBC cells are more sensitive to lexatumumab than the TRAIL-R1 agonist mapatumumab in vitro and in vivo (28), pointing to TRAIL-R2 as a promising therapeutic target in metastatic TNBC.

From a mechanism standpoint, we have shown that methionine depletion suppresses the protein levels of MAGED2, which provides a molecular link between methionine stress and enhanced TRAIL receptor sensitivity. Specifically, stably silencing MAGED2 mimics many of the effects of methionine depletion, including enhanced mRNA and cell surface expression of TRAIL receptors and increased sensitivity to TRAIL receptor agonists. However, methionine stress augments the cell surface expression of TRAIL-R2 to a greater extent than TRAIL-R1, while MAGED2 silencing enhances cell surface expression of both receptors, suggesting that additional proteins that are differentially regulated by methionine depletion contribute to its effects on TRAIL receptors. MAGED2 is a ubiquitously expressed member of the melanoma-associated antigen (MAGE)-II family that has been implicated in apoptosis-resistance by inhibiting TRAIL-R2 expression and/or antagonizing p53 function (36, 47, 48). Indeed, MAGED2 was previously identified as a negative regulator of TRAIL-R2 expression and TRAIL-induced apoptosis in melanoma cells (36). However, the mechanisms by which MAGED2 suppressed mRNA levels and cell surface expression of TRAIL-R2 were not delineated. Although we have demonstrated that methionine stress downregulates MAGED2 expression by a post-transcriptional mechanism, the details of this mechanism have yet to be elucidated. Furthermore, it remains to be determined how MAGED2 regulates TRAIL receptor expression. In contrast to the previous report (36), p53 is not required for the MAGED2-dependent effects on TRAIL-R2 expression in our experiments, as silencing MAGED2 in p53-mutant MDA-MB-231 TNBC cells suppressed TRAIL-R2 expression. Clearly, further studies are warranted to illuminate the molecular pathways linking methionine stress, reduced MAGED2 expression and enhanced TRAIL receptor levels. Nevertheless, our findings unequivocally point to the importance of MAGED2 and TRAIL-R2 in linking methionine stress to enhanced sensitivity to TRAIL receptor agonists.

In addition, our mechanistic observations may also be relevant to other nutritional interventions that have been reported to inhibit tumor growth, such as protein or caloric restriction, as these interventions result in decreased methionine intake (49). Indeed, protein restriction was recently reported to reduced cancer incidence in individuals aged 50–65 and inhibit tumor burden in murine models of cancer (50). Although serum IGF-1 levels were reduced by protein restriction in this study, it will be interesting to examine whether protein restriction sensitizes transformed cells to TRAIL agonists by increasing TRAIL-R2 expression. Such studies could lead to additional strategies to combine nutritional interventions and targeted therapies to improve clinical outcomes.

In summary, our results demonstrate that metabolic stress exposes distinctive vulnerabilities in transformed cells that can be targeted with rationally selected therapies. Specifically, methionine depletion exposes a targetable defect in TNBC cells by robustly increasing cell surface expression of TRAIL-R2 and rendering them more sensitive to TRAIL-R2 agonists in vitro and in vivo. Our findings provide proof-of-principle preclinical evidence to support a clinical trial combining dietary methionine restriction and proapoptotic TRAIL-R2 receptor agonists.

Supplementary Material

1
2
3
4
5

Translational Relevance.

Despite their potential tumor-selectivity and safety, proapoptotic TNF-related apoptosis-inducing ligand (TRAIL) receptor agonists, including recombinant TRAIL and humanized agonistic monoclonal antibodies, have been largely disappointing in clinical trials in patients with advanced malignancies. Here we report that methionine depletion enhances the sensitivity of triple (estrogen receptor, progesterone receptor and HER2)-negative breast cancer cells to lexatumumab, a proapoptotic humanized agonistic monoclonal antibody targeting TRAIL receptor-2 (TRAIL-R2). Methionine depletion augments lexatumumab-induced caspase activation and apoptosis by increasing TRAIL-R2 mRNA and cell surface expression. Notably, these effects were not observed in untransformed breast epithelial cells, suggesting that methionine stress selectively unmasks a targetable vulnerability in transformed cells. In addition, dietary methionine deprivation enhances the antitumor effects of lexatumumab in an orthotopic model of metastatic triple-negative breast cancer. Taken together, our results provide proof-of-principle preclinical evidence to support a clinical trial of dietary methionine restriction in combination with proapoptotic TRAIL-R2 agonists.

Acknowledgments

We are indebted to Mark Burkard and members of the Cryns lab for critical reading of the manuscript, Joshua Coon for use of the mass spectrometer, and Robin Humphreys for providing lexatumumab.

Grant support: Breast Cancer Research Foundation (VLC), Susan G. Komen for the Cure Postdoctoral Fellowship Award (DM), and P30CA 14520 University of Wisconsin Comprehensive Cancer Center core facility support.

Footnotes

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.

References

  • 1.Kreis W, Baker A, Ryan V, Bertasso A. Effect of nutritional and enzymatic methionine deprivation upon human normal and malignant cells in tissue culture. Cancer Res. 1980;40:634–41. [PubMed] [Google Scholar]
  • 2.Maddocks OD, Berkers CR, Mason SM, Zheng L, Blyth K, Gottlieb E, et al. Serine starvation induces stress and p53-dependent metabolic remodelling in cancer cells. Nature. 2013;493:542–6. doi: 10.1038/nature11743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Scott L, Lamb J, Smith S, Wheatley DN. Single amino acid (arginine) deprivation: rapid and selective death of cultured transformed and malignant cells. Br J Cancer. 2000;83:800–10. doi: 10.1054/bjoc.2000.1353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Sheen JH, Zoncu R, Kim D, Sabatini DM. Defective regulation of autophagy upon leucine deprivation reveals a targetable liability of human melanoma cells in vitro and in vivo. Cancer Cell. 2011;19:613–28. doi: 10.1016/j.ccr.2011.03.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Sugimura T, Birnbaum SM, Winitz M, Greenstein JP. Quantitative nutritional studies with water-soluble, chemically defined diets. VII. Nitrogen balance in normal and tumor-bearing rats following forced feeding. Arch Biochem Biophys. 1959;81:439–47. doi: 10.1016/0003-9861(59)90224-3. [DOI] [PubMed] [Google Scholar]
  • 6.Mecham JO, Rowitch D, Wallace CD, Stern PH, Hoffman RM. The metabolic defect of methionine dependence occurs frequently in human tumor cell lines. Biochem Bioph Res Co. 1983;117:429–34. doi: 10.1016/0006-291x(83)91218-4. [DOI] [PubMed] [Google Scholar]
  • 7.Guo HY, Herrera H, Groce A, Hoffman RM. Expression of the biochemical defect of methionine dependence in fresh patient tumors in primary histoculture. Cancer Res. 1993;53:2479–83. [PubMed] [Google Scholar]
  • 8.Hoshiya Y, Guo H, Kubota T, Inada T, Asanuma F, Yamada Y, et al. Human tumors are methionine dependent in vivo. Anticancer Res. 1995;15:717–8. [PubMed] [Google Scholar]
  • 9.Lu S, Hoestje SM, Choo E, Epner DE. Induction of caspase-dependent and -independent apoptosis in response to methionine restriction. Int J Oncol. 2003;22:415–20. [PubMed] [Google Scholar]
  • 10.Halpern BC, Clark BR, Hardy DN, Halpern RM, Smith RA. The effect of replacement of methionine by homocystine on survival of malignant and normal adult mammalian cells in culture. Proc Natl Acad Sci USA. 1974;71:1133–6. doi: 10.1073/pnas.71.4.1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Kreis W, Goodenow M. Methionine requirement and replacement by homocysteine in tissue cultures of selected rodent and human malignant and normal cells. Cancer Res. 1978;38:2259–62. [PubMed] [Google Scholar]
  • 12.Tan Y, Zavala J, Sr, Xu M, Zavala J, Jr, Hoffman RM. Serum methionine depletion without side effects by methioninase in metastatic breast cancer patients. Anticancer Res. 1996;16:3937–42. [PubMed] [Google Scholar]
  • 13.Yoshioka T, Wada T, Uchida N, Maki H, Yoshida H, Ide N, et al. Anticancer efficacy in vivo and in vitro, synergy with 5-fluorouracil, and safety of recombinant methioninase. Cancer Res. 1998;58:2583–7. [PubMed] [Google Scholar]
  • 14.Stern PH, Hoffman RM. Enhanced in vitro selective toxicity of chemotherapeutic agents for human cancer cells based on a metabolic defect. J Natl Cancer I. 1986;76:629–39. doi: 10.1093/jnci/76.4.629. [DOI] [PubMed] [Google Scholar]
  • 15.Tan Y, Sun X, Xu M, Tan X, Sasson A, Rashidi B, et al. Efficacy of recombinant methioninase in combination with cisplatin on human colon tumors in nude mice. Clin Cancer Res. 1999;5:2157–63. [PubMed] [Google Scholar]
  • 16.Poirson-Bichat F, Goncalves RA, Miccoli L, Dutrillaux B, Poupon MF. Methionine depletion enhances the antitumoral efficacy of cytotoxic agents in drug-resistant human tumor xenografts. Clin Cancer Res. 2000;6:643–53. [PubMed] [Google Scholar]
  • 17.Najim N, Podmore ID, McGown A, Estlin EJ. Methionine restriction reduces the chemosensitivity of central nervous system tumour cell lines. Anticancer Res. 2009;29:3103–8. [PubMed] [Google Scholar]
  • 18.Hoffman RM, Erbe RW. High in vivo rates of methionine biosynthesis in transformed human and malignant rat cells auxotrophic for methionine. Proc Natl Acad Sci USA. 1976;73:1523–7. doi: 10.1073/pnas.73.5.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Stern PH, Wallace CD, Hoffman RM. Altered methionine metabolism occurs in all members of a set of diverse human tumor cell lines. J Cell Physiol. 1984;119:29–34. doi: 10.1002/jcp.1041190106. [DOI] [PubMed] [Google Scholar]
  • 20.Cellarier E, Durando X, Vasson MP, Farges MC, Demiden A, Maurizis JC, et al. Methionine dependency and cancer treatment. Cancer Treat Rev. 2003;29:489–99. doi: 10.1016/s0305-7372(03)00118-x. [DOI] [PubMed] [Google Scholar]
  • 21.Kokkinakis DM, Liu X, Chada S, Ahmed MM, Shareef MM, Singha UK, et al. Modulation of gene expression in human central nervous system tumors under methionine deprivation-induced stress. Cancer Res. 2004;64:7513–25. doi: 10.1158/0008-5472.CAN-04-0592. [DOI] [PubMed] [Google Scholar]
  • 22.Ashkenazi A. Directing cancer cells to self-destruct with pro-apoptotic receptor agonists. Nat Rev Drug Discov. 2008;7:1001–12. doi: 10.1038/nrd2637. [DOI] [PubMed] [Google Scholar]
  • 23.Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA, Marsters SA, et al. Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest. 1999;104:155–62. doi: 10.1172/JCI6926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med. 1999;5:157–63. doi: 10.1038/5517. [DOI] [PubMed] [Google Scholar]
  • 25.Buchsbaum DJ, Zhou T, Grizzle WE, Oliver PG, Hammond CJ, Zhang S, et al. Antitumor efficacy of TRA-8 anti-DR5 monoclonal antibody alone or in combination with chemotherapy and/or radiation therapy in a human breast cancer model. Clin Cancer Res. 2003;9:3731–41. [PubMed] [Google Scholar]
  • 26.Lu M, Strohecker A, Chen F, Kwan T, Bosman J, Jordan VC, et al. Aspirin sensitizes cancer cells to TRAIL-induced apoptosis by reducing survivin levels. Clin Cancer Res. 2008;14:3168–76. doi: 10.1158/1078-0432.CCR-07-4362. [DOI] [PubMed] [Google Scholar]
  • 27.Marini P, Denzinger S, Schiller D, Kauder S, Welz S, Humphreys R, et al. Combined treatment of colorectal tumours with agonistic TRAIL receptor antibodies HGS-ETR1 and HGS-ETR2 and radiotherapy: enhanced effects in vitro and dose-dependent growth delay in vivo. Oncogene. 2006;25:5145–54. doi: 10.1038/sj.onc.1209516. [DOI] [PubMed] [Google Scholar]
  • 28.Malin D, Chen F, Schiller C, Koblinski J, Cryns VL. Enhanced metastasis suppression by targeting TRAIL receptor 2 in a murine model of triple-negative breast cancer. Clin Cancer Res. 2011;17:5005–15. doi: 10.1158/1078-0432.CCR-11-0099. [DOI] [PubMed] [Google Scholar]
  • 29.Camidge DR, Herbst RS, Gordon MS, Eckhardt SG, Kurzrock R, Durbin B, et al. A phase I safety and pharmacokinetic study of the death receptor 5 agonistic antibody PRO95780 in patients with advanced malignancies. Clin Cancer Res. 2010;16:1256–63. doi: 10.1158/1078-0432.CCR-09-1267. [DOI] [PubMed] [Google Scholar]
  • 30.Herbst RS, Eckhardt SG, Kurzrock R, Ebbinghaus S, O’Dwyer PJ, Gordon MS, et al. Phase I dose-escalation study of recombinant human Apo2L/TRAIL, a dual proapoptotic receptor agonist, in patients with advanced cancer. J Clin Oncol. 2010;28:2839–46. doi: 10.1200/JCO.2009.25.1991. [DOI] [PubMed] [Google Scholar]
  • 31.Herbst RS, Kurzrock R, Hong DS, Valdivieso M, Hsu CP, Goyal L, et al. A first-inhuman study of conatumumab in adult patients with advanced solid tumors. Clin Cancer Res. 2010;16:5883–91. doi: 10.1158/1078-0432.CCR-10-0631. [DOI] [PubMed] [Google Scholar]
  • 32.Merchant MS, Geller JI, Baird K, Chou AJ, Galli S, Charles A, et al. Phase I trial and pharmacokinetic study of lexatumumab in pediatric patients with solid tumors. J Clin Oncol. 2012;30:4141–7. doi: 10.1200/JCO.2012.44.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Trarbach T, Moehler M, Heinemann V, Kohne CH, Przyborek M, Schulz C, et al. Phase II trial of mapatumumab, a fully human agonistic monoclonal antibody that targets and activates the tumour necrosis factor apoptosis-inducing ligand receptor-1 (TRAIL-R1), in patients with refractory colorectal cancer. Br J Cancer. 2010;102:506–12. doi: 10.1038/sj.bjc.6605507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Wakelee HA, Patnaik A, Sikic BI, Mita M, Fox NL, Miceli R, et al. Phase I and pharmacokinetic study of lexatumumab (HGS-ETR2) given every 2 weeks in patients with advanced solid tumors. Ann Oncol. 2010;21:376–81. doi: 10.1093/annonc/mdp292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Dimberg LY, Anderson CK, Camidge R, Behbakht K, Thorburn A, Ford HL. On the TRAIL to successful cancer therapy? Predicting and counteracting resistance against TRAIL-based therapeutics. Oncogene. 2013;32:1341–50. doi: 10.1038/onc.2012.164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Tseng HY, Chen LH, Ye Y, Tay KH, Jiang CC, Guo ST, et al. The melanoma-associated antigen MAGE-D2 suppresses TRAIL receptor 2 and protects against TRAIL-induced apoptosis in human melanoma cells. Carcinogenesis. 2012;33:1871–81. doi: 10.1093/carcin/bgs236. [DOI] [PubMed] [Google Scholar]
  • 37.Malin D, Strekalova E, Petrovic V, Deal AM, Al Ahmad A, Adamo B, et al. αB-crystallin: a novel regulator of breast cancer metastasis to the brain. Clin Cancer Res. 2014;20:56–67. doi: 10.1158/1078-0432.CCR-13-1255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Moyano JV, Evans JR, Chen F, Lu M, Werner ME, Yehiely F, et al. αB-crystallin is a novel oncoprotein that predicts poor clinical outcome in breast cancer. J Clin Invest. 2006;116:261–70. doi: 10.1172/JCI25888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Lu M, Kwan T, Yu C, Chen F, Freedman B, Schafer JM, et al. Peroxisome proliferator-activated receptor γ agonists promote TRAIL-induced apoptosis by reducing survivin levels via cyclin D3 repression and cell cycle arrest. J Biol Chem. 2005;280:6742–51. doi: 10.1074/jbc.M411519200. [DOI] [PubMed] [Google Scholar]
  • 40.Guadamillas MC, Cerezo A, Del Pozo MA. Overcoming anoikis--pathways to anchorage-independent growth in cancer. J Cell Sci. 2011;124:3189–97. doi: 10.1242/jcs.072165. [DOI] [PubMed] [Google Scholar]
  • 41.Guo H, Lishko VK, Herrera H, Groce A, Kubota T, Hoffman RM. Therapeutic tumor-specific cell cycle block induced by methionine starvation in vivo. Cancer Res. 1993;53:5676–9. [PubMed] [Google Scholar]
  • 42.Takeda K, Hayakawa Y, Smyth MJ, Kayagaki N, Yamaguchi N, Kakuta S, et al. Involvement of tumor necrosis factor-related apoptosis-inducing ligand in surveillance of tumor metastasis by liver natural killer cells. Nat Med. 2001;7:94–100. doi: 10.1038/83416. [DOI] [PubMed] [Google Scholar]
  • 43.Grosse-Wilde A, Voloshanenko O, Bailey SL, Longton GM, Schaefer U, Csernok AI, et al. TRAIL-R deficiency in mice enhances lymph node metastasis without affecting primary tumor development. J Clin Invest. 2008;118:100–10. doi: 10.1172/JCI33061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Epner DE, Morrow S, Wilcox M, Houghton JL. Nutrient intake and nutritional indexes in adults with metastatic cancer on a phase I clinical trial of dietary methionine restriction. Nutr Cancer. 2002;42:158–66. doi: 10.1207/S15327914NC422_2. [DOI] [PubMed] [Google Scholar]
  • 45.Durando X, Thivat E, Farges MC, Cellarier E, D’Incan M, Demidem A, et al. Optimal methionine-free diet duration for nitrourea treatment: a Phase I clinical trial. Nutr Cancer. 2008;60:23–30. doi: 10.1080/01635580701525877. [DOI] [PubMed] [Google Scholar]
  • 46.Thivat E, Farges MC, Bacin F, D’Incan M, Mouret-Reynier MA, Cellarier E, et al. Phase II trial of the association of a methionine-free diet with cystemustine therapy in melanoma and glioma. Anticancer Res. 2009;29:5235–40. [PubMed] [Google Scholar]
  • 47.Papageorgio C, Brachmann R, Zeng J, Culverhouse R, Zhang W, McLeod H. MAGED2: a novel p53-dissociator. Int J Oncol. 2007;31:1205–11. [PubMed] [Google Scholar]
  • 48.Sang M, Wang L, Ding C, Zhou X, Wang B, Wang L, et al. Melanoma-associated antigen genes - an update. Cancer Lett. 2011;302:85–90. doi: 10.1016/j.canlet.2010.10.021. [DOI] [PubMed] [Google Scholar]
  • 49.Mirzaei H, Suarez JA, Longo VD. Protein and amino acid restriction, aging and disease: from yeast to humans. Trends Endocrinol Metabol. 2014;25:558–66. doi: 10.1016/j.tem.2014.07.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Levine ME, Suarez JA, Brandhorst S, Balasubramanian P, Cheng CW, Madia F, et al. Low protein intake is associated with a major reduction in IGF-1, cancer, and overall mortality in the 65 and younger but not older population. Cell Metabolism. 2014;19:407–17. doi: 10.1016/j.cmet.2014.02.006. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS668702-supplement-1.docx (29.8KB, docx)
2
NIHMS668702-supplement-2.pptx (52.2KB, pptx)
3
NIHMS668702-supplement-3.xlsx (40.6KB, xlsx)
4
NIHMS668702-supplement-4.pptx (44.1KB, pptx)
5
NIHMS668702-supplement-5.pptx (549.1KB, pptx)

RESOURCES