DNA methylation status of key cell-cycle regulators such as CDKNA2/p16 and CCNA1 correlates with treatment response to doxorubicin and 5-fluorouracil in locally advanced breast tumors

Abstract

Purpose: To explore alterations in gene promoter methylation as a potential cause of acquired drug resistance to doxorubicin or combined treatment with 5-fluorouracil and mitomycin C in human breast cancers.

Experimental design: Paired tumor samples from locally advanced breast cancer patients treated with doxorubicin and 5-fluorouracil-mitomycin C were used in the genome-wide DNA methylation analysis as discovery cohort. An enlarged cohort from the same two prospective studies as those in the discovery cohort was used as a validation set in pyrosequencing analysis.

Results: A total of 469 genes were differentially methylated after treatment with doxorubicin and revealed a significant association with canonical pathways enriched for immune cell response and cell-cycle regulating genes including CDKN2A, CCND2, CCNA1, which were also associated to treatment response. Treatment with FUMI resulted in 343 differentially methylated genes representing canonical pathways such as retinoate biosynthesis, gαi signaling, and LXR/RXR activation. Despite the clearly different genes and pathways involved in the metabolism and therapeutic effect of both drugs, 46 genes were differentially methylated before and after treatment with both doxorubicin and FUMI. DNA methylation profiles in genes such as BRCA1, FOXC1, and IGFBP3, and most notably repetitive elements like ALU and LINE1, were associated with TP53 mutations status.

Conclusion: We identified and validated key cell-cycle regulators differentially methylated before and after neoadjuvant chemotherapy such as CDKN2A and CCNA1 and reported that methylation patterns of these genes may be potential predictive markers to anthracycline/mitomycine sensitivity.