Reversion of methionine addiction of osteosarcoma cells to methionine independence results in loss of malignancy, modulation of the epithelial-mesenchymal phenotype and alteration of histone-H3 lysine-methylation

Abstract

Methionine addiction, a fundamental and general hallmark of cancer, known as the Hoffman Effect, is due to altered use of methionine for increased and aberrant transmethylation reactions. However, the linkage of methionine addiction and malignancy of cancer cells is incompletely understood. An isogenic pair of methionine-addicted parental osteosarcoma cells and their rare methionine-independent revertant cells enabled us to compare them for malignancy, their epithelial-mesenchymal phenotype, and pattern of histone-H3 lysine-methylation. Methionine-independent revertant 143B osteosarcoma cells (143B-R) were selected from methionine-addicted parental cells (143B-P) by their chronic growth in low-methionine culture medium for 4 passages, which was depleted of methionine by recombinant methioninase (rMETase). Cell-migration capacity was compared with a wound-healing assay and invasion capability was compared with a transwell assay in 143B-P and 143B-R cells in vitro. Tumor growth and metastatic potential were compared after orthotopic cell-injection into the tibia bone of nude mice in vivo. Epithelial-mesenchymal phenotypic expression and the status of H3 lysine-methylation were determined with western immunoblotting. 143B-P cells had an IC50 of 0.20 U/ml and 143B-R cells had an IC50 of 0.68 U/ml for treatment with rMETase, demonstrating that 143B-R cells had regained the ability to grow in low methionine conditions. 143B-R cells had reduced cell migration and invasion capability in vitro, formed much smaller tumors than 143B-P cells and lost metastatic potential in vivo, indicating loss of malignancy in 143B-R cells. 143B-R cells showed gain of the epithelial marker, ZO-1 and loss of mesenchymal markers, vimentin, Snail, and Slug and, an increase of histone H3K9me3 and H3K27me3 methylation and a decrease of H3K4me3, H3K36me3, and H3K79me3 methylation, along with their loss of malignancy. These results suggest that shifting the balance in histone methylases might be a way to decrease the malignant potential of cells. The present results demonstrate the rationale to target methionine addiction for improved sarcoma therapy.

Oncogenes and Methionine Addiction of Cancer: Role ofc-MYC

“…Female athymic nu/nu nude mice (4-6 weeks old) (AntiCancer, Inc., San Diego, CA, USA) were used in the present study. All mice were bred and maintained as previously reported (12), under Assurance Number A3873-1, with an Institutional Animal Care and Use Committee (IACUC) protocol, following the National Institutes of Health (NIH) Guide for the Care and Use of Animals. For all surgical procedures, a cocktail of anesthetics and analgesics (ketamine: 20 mg/kg, xylazine: 15.2 mg/kg, acepromazine maleate: 0.48 mg/kg) was used, in order to minimize animal distress.…”

“…We subsequently reported that reversion of methionineaddicted osteosarcoma cells to methionine-independence reduced malignancy, along with reduced epithelial-tomesenchymal transition (12).…”

“…Methionine addiction, which includes folate addiction (3), is termed the Hoffman effect (2). It appears all cancer types are addicted to methionine (7)(8)(9)(10)(11)(12). Methionine addiction is due to overuse of methionine for transmethylation reactions (7,(9)(10)(11)(12)(13).…”

“…It appears all cancer types are addicted to methionine (7)(8)(9)(10)(11)(12). Methionine addiction is due to overuse of methionine for transmethylation reactions (7,(9)(10)(11)(12)(13).…”

“…Since then, the role of other oncogenes in methionine addiction has not been investigated. It has been previously reported that methionine-independent revertant cells, which were selected from methionine-addicted parental cancer cells by continuous culture in low-methionine medium, have reduced malignancy (8,(10)(11)(12)(15)(16)(17), in part due to changes in methylation of histone-H3 lysine marks (7,(9)(10)(11)(12).…”