| We report that DNA methyltransferase 1 (DNMT1) expression is dysregulated in breast cancer. The elevated protein levels are not a result of increased mRNA levels, but rather an increase in protein half-life. We found that DNMT1 protein levels were elevated in breast cancer tissues and in MCF-7 breast cancer cells relative to normal Human Mammary Epithelial Cells (HMECs) without a concomitant increase in DNMT1 mRNA or proliferative fraction. While DNMT1 mRNA levels were properly S-phase regulated in both cell types, DNMT1 protein levels did not follow S-phase fraction in MCF-7 cells. Rather, an increase in DNMT1 protein stability was found for MCF-7 cells relative to HMECs, and a destruction domain was mapped to the N-terminal 120 amino acids of DNMT1 which was required for its proper ubiquitination and degradation in HMECs. Furthermore, overexpression of DNMT1 with this deleted destruction domain in HMECs resulted in significantly increased genomic 5-methylcytosine levels relative to overexpression of the full-length protein. The regulation of DNMT1 destruction via this domain may be dysfunctional in cancer cells leading to subsequent cytosine hypermethylation in the genome. Depletion of CDC20 and FZR1, two substrate recognition adaptor components of the Anaphase Promoting Complex resulted in stabilization of DNMT1 that was partially dependent on the N-terminal destruction domain, implicating this cell cycle regulating ubiquitin ligase complex in the destruction of DNMT1. MAD2, an inhibitor of CDC20, was shown to stabilize DNMT1 levels, and overexpression of MAD2, a consequence of RB pathway dysregulation, was shown to correlate with impaired G1-phase DNMT1 destruction. This suggests that RB pathway inactivation, a common dysfunction in cancer cells, may be the underlying cause of DNMT1 dysregulation. |
