Abstract 3896: Alteration of malignancy and histone-H3 lysine-methylation status in osteosarcoma cells which acquire methotrexate resistance in vitro
Authors: Aoki Yusuke , Tome Yasunori , Oshiro Hiromichi , Katsuki Ryo , Mizuta Kohei , Nishida Kotaro , Hoffman Robert M. 
Published: April 2023

Since methotrexate (MTX) resistance of osteosarcoma is a frequent cause of poor prognosis, new treatment strategies are needed to overcome this recalcitrant problem. The present study investigated if MTX resistance increased malignancy of osteosarcoma cells and its possible mechanism, via histone-H3 lysine-methylation status. We also propose a new therapeutic strategy for MTX resistant osteosarcoma. MTX resistant 143B osteosarcoma cells (143B-MTXR) were established from 143B parental osteosarcoma cells (143B-P) by culturing the cells with increasing concentrations of MTX stepwise (0.04 μM to 100 μM) for 12 months. The degree of malignancy of the isogenic pair of 143B-MTXR and 143B-P cells was compared by testing clonogenic capacity in cell culture, on both plastic and soft agar. Malignancy was also compared by tumor growth in orthotopic xenograft mouse models by implantation of 2.5 × 105 cells in the tibia of nude mice. Histone-H3 lysine-methylation status was determined by Western immunoblotting. 143B-MTXR and 143B-P cells were also tested for methionine addiction by sensitivity to recombinant methioninase (rMETase). Welch’s t-test was performed to statistically evaluate results and a probability value ≤ 0.05 was defined as statistically-significant difference. The mouse studies were approved by Institutional Animal Care and Use Committee of AntiCancer Inc. After 12 months selection in increasing concentrations of MTX, we isolated 143B-MTXR cells, which had an MTX IC50 of 384 μM compared to 143B-P cells, which had an IC50 of 0.27 μM, demonstrating that the 143B-MTXR cells acquired a 1422-fold resistance to MTX. In cell culture, 143B-MTXR cells had reduced clonogenic capacity on plastic and in soft agar (P &lt; 0.01), indicating reduction in malignancy. In orthotopic xenograft nude-mouse studies, 143B-MTXR cells formed much smaller tumors than 143B-P cells (P &lt; 0.01), further indicating that 143B-MTXR cells lost malignancy. 143B-MTXR cells showed an increase of histone H3K9me3 (P = 0.01), H3K27me3 (P &lt; 0.01), and H3K79me3 (P &lt; 0.01) methylation, which may account in part for their decreased malignancy. However, there was no difference in methylation of H3K4me3 (P = 0.12) or H3K36me3 (P = 0.29). The 143B-MTXR cells had an IC50 of 0.358 U/ml for rMETase, similar to an IC50 of 0.38 U/ml for 143B-P cells, indicating the 143B-MTXR cells did not lose their methionine addiction. The increase in histone H3K9me3, H3K27me3, and H3K79me3 shown in the present study in 143B-MTXR cells suggested potential biomarkers of methotrexate-resistance. MTX resistant osteosarcoma may be treated by methionine restriction with rMETase, as the MTX-resistant osteosarcoma cells are highly methionine addicted.

Citation Format: Yusuke Aoki, Yasunori Tome, Hiromichi Oshiro, Ryo Katsuki, Kohei Mizuta, Kotaro Nishida, Robert M. Hoffman. Alteration of malignancy and histone-H3 lysine-methylation status in osteosarcoma cells which acquire methotrexate resistance in vitro. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3896.