Quantitative proteomics reveals mechanisms of methotrexate resistance (mtx-r) in gestational trophoblastic neoplasia (gtn) and other cancer types

Patients with low-risk gestational trophoblastic neoplasia (GTN) after a molar/non-molar pregnancy are mostly treated with methotrexate (MTX), but many develop resistance (MTX-R), requiring alternative or more toxic combination therapies to achieve remission. Understanding the molecular mechanisms of MTX-R is crucial for improving treatment. This study employed proteomics profiling of patient tissue samples to explore alterations linked to MTX-R in patients and human choriocarcinoma cell lines. Bioinformatics analysis identified the Serine Synthesis Pathway (SSP) as significantly downregulated in MTX-R patients and the resistant choriocarcinoma cell line JEG3-R. Phosphoserine aminotransferase 1 (PSAT1), a key SSP enzyme, emerged as a potential prognostic marker for MTX sensitivity in a small patient cohort following Immunohistochemistry (IHC) analysis. Functional studies showed that simultaneous serine deprivation and inhibition of de novo serine synthesis increased MTX resistance in choriocarcinoma cells. Conversely, overexpression of PHGDH and PSAT1, combined with serine and glycine depletion, reversed resistance in JEG3-R cells, restoring MTX sensitivity. Further experimentation revealed that MTX-R cells exhibit enhanced detoxification capacity, redirecting serine towards glutathione synthesis while downregulating energy-demanding processes like proliferation. This adaptation helps resistant cells mitigate MTX-induced oxidative stress contributing to MTX-R. Investigation into the underlying mechanisms suggested that activation of the Integrated Stress Response (ISR) pathway in JEG3-R cells facilitates translational blockage of non-essential pathways while promoting survival mechanisms such as autophagy. Moreover, dose-specific inhibition of the ISR managed to sensitize JEG3-R cells to MTX indicating a potential therapeutic strategy in the resistant choriocarcinoma. Finally, SSP downregulation was examined in MTX-sensitive and MTX-resistant breast, colon, and osteosarcoma cell lines. Notably, four of seven MTX-R lines displayed similar SSP downregulation as choriocarcinoma MTX-R cells, reinforcing the role of serine metabolism in MTX response. These findings highlight the potential of SSP as a target for overcoming MTX-R and advancing clinical research.