2. Academic Validation
  3. Reduced Malignancy of Super Methotrexate-resistant Osteosarcoma Cells With Dihydrofolate Reductase Amplification Despite Paradoxical Gain of Oncogenic PI3K/AKT/mTOR and c-MYC expression

Reduced Malignancy of Super Methotrexate-resistant Osteosarcoma Cells With Dihydrofolate Reductase Amplification Despite Paradoxical Gain of Oncogenic PI3K/AKT/mTOR and c-MYC expression

  • Anticancer Res. 2024 Jul;44(7):2787-2792. doi: 10.21873/anticanres.17090.
Yusuke Aoki 1 2 3,  Yutaro Kubota 4 2,  Noriyuki Masaki 4 2,  Koya Obara 4 2,  Yasunori Tome 5,  Michael Bouvet 2,  Kotaro Nishida 3,  Robert M Hoffman 6 2
Affiliations

Affiliations

  • 1 AntiCancer Inc, San Diego, CA, U.S.A.; yaoki0630@gmail.com.
  • 2 Department of Surgery, University of California, San Diego, La Jolla, CA, U.S.A.
  • 3 Department of Orthopedic Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan.
  • 4 AntiCancer Inc, San Diego, CA, U.S.A.
  • 5 Department of Orthopedic Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan yastome@med.u-ryukyu.ac.jp.
  • 6 AntiCancer Inc, San Diego, CA, U.S.A.; all@anticancer.com.
PMID: 38925854 DOI: 10.21873/anticanres.17090
Abstract

Background/aim: Methotrexate (MTX) resistance in osteosarcoma leads to a very poor prognosis. In the present study, in order to further understand the basis and ramifications of MTX resistance in osteosarcoma, we selected an osteosarcoma cell line that has a 5,500-fold-increased MTX IC50 Materials and Methods: The super MTX-resistant 143B osteosarcoma cells (143B-MTXSR) were selected from MTX-sensitive parental human 143B osteosarcoma cells (143B-P) by continuous culture with step-wise increased amounts of MTX. To compare the malignancy of 143B-MTXSR and 143B-P, colony-formation capacity was compared with clonogenic assays on plastic and in soft agar. In addition, tumor growth was compared with orthotopic xenograft mouse models of osteosarcoma. Expression of dihydrofolate reductase (DHFR), phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), and myelocytomatosis oncogene (MYC) was examined with western immunoblotting and compared in 143B-MTXSR and 143B-P cells.

Results: 143B-MTXSR had a 5,500-fold increase in the MTX IC50 compared to the parental 143B-P cells. Expression of DHFR was increased 10-fold in 143B-MTXSR compared to 143B-P (p<0.01). 143B-MTXSR cells had reduced colony-formation capacity on plastic (p=0.032) and in soft agar (p<0.01) compared to 143B-P and reduced tumor growth in orthotopic xenograft mouse models (p<0.001). These results demonstrate that 143B-MTXSR had reduced malignancy. 143B-MTXSR also showed an increased expression of PI3K (p<0.01), phosphorylated (activated) Akt (p=0.031), phosphorylated mTOR (p=0.043), and c-Myc (p=0.024) compared to 143B-P.

Conclusion: The present study demonstrates that the increased expression of DHFR, PI3K/Akt/mTOR and c-Myc appears to be linked to super MTX resistance and, paradoxically, to reduced malignancy. The present results suggest that DHFR may be a powerful tumor suppressor when highly amplified.

Keywords

AKT; DHFR; Osteosarcoma; PI3K; c-MYC; mTOR; malignancy; methotrexate; nude mice; resistance; tumor suppressor.

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