Modulation of dietary methionine intake elicits potent, yet distinct, anticancer effects on primary versus metastatic tumors

Abstract

Methionine dependency describes the characteristic rapid in vitro death of most tumor cells in the absence of methionine. Combining chemotherapy with dietary methionine deprivation [methionine-deficient diet (MDD)] at tolerable levels has vast potential in tumor treatment; however, it is limited by MDD-induced toxicity during extended deprivation. Recent advances in imaging and irradiation delivery have created the field of stereotactic body radiotherapy (SBRT), where fewer large-dose fractions delivered in less time result in increased local-tumor control, which could be maximally synergistic with an MDD short course. Identification of the lowest effective methionine dietary intake not associated with toxicity will further enhance the cancer therapy potential. In this study, we investigated the effects of MDD and methionine-restricted diet (MRD) in primary and metastatic melanoma models in combination with radiotherapy (RT). In vitro, MDD dose-dependently sensitized mouse and human melanoma cell lines to RT. In vivo in mice, MDD substantially potentiated the effects of RT by a significant delay in tumor growth, in comparison with administering MDD or RT alone. The antitumor effects of an MDD/RT approach were due to effects on one-carbon metabolism, resulting in impaired methionine biotransformation via downregulation of Mat2a, which encodes methionine adenosyltransferase 2A. Furthermore, and probably most importantly, MDD and MRD substantially diminished metastatic potential; the antitumor MRD effects were not associated with toxicity to normal tissue. Our findings suggest that modulation of methionine intake holds substantial promise for use with short-course SBRT for cancer treatment.

Figure 1.

Effects of methionine deprivation on melanoma cells in vitro and in vivo. Wound healing assay in (A) B16-F10.1 and (B) A375-MA1 cells under various methionine concentrations. Clonogenic survival of (C) B16-F10.1 and (D) A375-MA1cells under various methionine-restriction conditions after exposure to X-rays. (E) Schematic representation of experimental setup for primary melanoma model. (F) Effects of methionine deprivation on experimental B16-F10.1 melanoma model in vivo. Data are presented as means ± standard error of the mean.

Figure 2.

Role of one-carbon metabolism in tumor radiosensitization induced by methionine deprivation. (A) Schematic representation of methionine cycle (5-meTHF, 5-methyltetrahydrofolate; Bhmt, betaine-homocysteine S-methyltransferase; Cbs, cystathionine-β-synthase; DMG, dimethylglycine; Dnmt, DNA methyltransferase; Mat, methionine adenosyltransferase; Mtr, methionine synthase; Sahh, s-adenosyl-l-homocysteine hydrolase; THF, tetrahydrofolate. (B) Gene expression analysis of critical players in one-carbon metabolism. (C) Abundance of Mat2a protein relative to TATA-binding protein (TBP) in B16-F10.1 cells in vivo. MAD: lanes 1–3, MDD: lanes 4–6, MAD/IR (10 Gy): lanes 7–9, MDD/IR (10 Gy): lanes 10–12. (D) Experimental set-up with administration of FIDAS-5. (E) Synergistic effects of FIDAS-5 and MDD on melanoma in vivo.

Figure 3.

Effects of modulation of methionine dietary intake on distant metastases. (A) Microphotograph of the large metastasis confined to pulmonary vein that failed to extravasate (×4 and ×20 magnification, H and E staining). (B) Photograph of pulmonary superficial metastases. (C) Pulmonary superficial metastasis counts. (D) Microphotographs of pulmonary metastases (H and E staining). (E) Survival of mice fed the three diets (MAD, MDD, MRD) after initiation of lung metastasis (i.e. 3 weeks after injection of B16-F10.1 cells via tail vein).

Figure 4.

RNA sequencing analysis of experimental pulmonary metastases. (A) Overlap of genes with expression that was significantly different in mice in the MDD or MRD group than in the MAD group. (B) Expression levels of the 22 genes common to MDD and MRD groups (from panel A). (C) GO terms significantly enriched in MDD and MRD groups. Bars indicate −log of the P-value for each specific GO term. Exact matches between the diets are indicated with dark blue bars, and close matches are in light blue.

Figure 5.

Methionine deprivation–associated normal tissue toxicity. (A) Body-weight dynamics in mice in experimental B16-F10.1 primary melanoma (IR: to 10 Gy of X-rays). (B) Microphotograph showing the features of initial steatosis in the livers of mice fed MDD (magnification ×20, H and E staining). (C) Body-weight dynamics in B16-F10.1 pulmonary metastasis model. (D) Microphotograph of healthy mouse liver after 35 days of MRD (magnification ×20, H and E staining).