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Updated: Jun 28, 2025
Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms
Published on: December 9, 2015
Dynamic genomic changes in methotrexate-resistant human cancer cell lines beyond DHFR amplification suggest potential
Xiang-Ning Meng1,2, Jin-Fa Ma1,2, Yang-He Liu1,2
1Key laboratory of preservation of human genetic resources and disease control in China (Harbin Medical University), Ministry of Education, Harbin, 150081, China.
Background:
Although DHFR gene amplification has long been known as a major mechanism for methotrexate (MTX) resistance in cancer, the early changes and detailed development of the resistance are not yet fully understood.
Methods:
We performed genomic, transcriptional and proteomic analyses of human colon cancer cells with sequentially increasing levels of MTX-resistance.
Results:
The genomic amplification evolved in three phases (pre-amplification, homogenously staining region (HSR) and extrachromosomal DNA (ecDNA)). We confirm that genomic amplification and increased expression of DHFR, with formation of HSRs and especially ecDNAs, is the major driver of resistance. However, DHFR did not play a detectable role in the early phase. In the late phase (ecDNA), increase in FAM151B protein level may also have an important role by decreasing sensitivity to MTX. In addition, although MSH3 and ZFYVE16 may be subject to different posttranscriptional regulations and therefore protein expressions are decreased in ecDNA stages compared to HSR stages, they still play important roles in MTX resistance.
Conclusion:
The study provides a detailed evolutionary trajectory of MTX-resistance and identifies new targets, especially ecDNAs, which could help to prevent drug resistance. It also presents a proof-of-principal approach which could be applied to other cancer drug resistance studies.
Insights
Methotrexate resistance in cancer develops through DHFR gene amplification in distinct phases. Extrachromosomal DNA (ecDNA) formation is a key driver, with FAM151B, MSH3, and ZFYVE16 also influencing resistance.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Dihydrofolate reductase (DHFR) gene amplification is a known mechanism of methotrexate (MTX) resistance in cancer.
- The early stages and detailed progression of MTX resistance remain incompletely understood.
Purpose of the Study:
- To elucidate the evolutionary trajectory of MTX resistance in colon cancer cells.
- To identify key genetic and proteomic changes driving resistance development.
- To uncover potential new targets for overcoming MTX resistance.
Main Methods:
- Genomic, transcriptional, and proteomic analyses were conducted.
- Human colon cancer cells were subjected to sequentially increasing levels of MTX.
- Comparative analysis across different resistance phases was performed.
Main Results:
- MTX resistance evolved through three genomic amplification phases: pre-amplification, homogenously staining regions (HSRs), and extrachromosomal DNA (ecDNA).
- DHFR amplification and increased expression were confirmed as major drivers, but DHFR was not critical in the early resistance phase.
- FAM151B protein increase in the ecDNA phase may reduce MTX sensitivity, while MSH3 and ZFYVE16 play roles despite altered protein expression.
Conclusions:
- The study details the evolutionary path of MTX resistance, highlighting ecDNAs as significant targets.
- FAM151B, MSH3, and ZFYVE16 are identified as potentially important factors in later stages of resistance.
- The findings offer insights into preventing or overcoming cancer drug resistance and suggest a model for studying other resistance mechanisms.
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