Ubiquitination and Degradation of Ribonucleotide Reductase M1 by the Polycomb Group Proteins RNF2 and Bmi1 and Cellular Response to Gemcitabine

Abstract

Ribonucleotide reductase M1 (RRM1) is required for mammalian deoxyribonucleotide (dNTP) metabolism. It is the primary target of the antimetabolite drug gemcitabine, which is among the most efficacious and most widely used cancer therapeutics. Gemcitabine directly binds to RRM1 and irreversibly inactivates ribonucleotide reductase. Intra-tumoral RRM1 levels are predictive of gemcitabine’s therapeutic efficacy. The mechanisms that regulate intracellular RRM1 levels are largely unknown. Here, we identified the E3 ubiquitin-protein ligases RNF2 and Bmi1 to associate with RRM1 with subsequent poly-ubiquitination at either position 48 or 63 of ubiquitin. The lysine residues 224 and 548 of RRM1 were identified as major ubiquitination sites. We show that ubiquitinated RRM1 undergoes proteasome-mediated degradation and that targeted post-transcriptional silencing of RNF2 and Bmi1 results in increased RRM1 levels and resistance to gemcitabine. Immunohistochemical analyses of 187 early-stage lung cancer tumor specimens revealed a statistically significant co-expression of RRM1 and Bmi1. We were unable to identify suitable reagents for in situ quantification of RNF2. Our findings suggest that Bmi1 and possibly RNF2 may be attractive biomarkers of gemcitabine resistance in the context of RRM1 expression. They also provide novel information for the rational design of gemcitabine-proteasome inhibitor combination therapies, which so far have been unsuccessful if given to patients without taking the molecular context into account.

The MEK1/2 Inhibitor Pimasertib Enhances Gemcitabine Efficacy in Pancreatic Cancer Models by Altering Ribonucleotide Reductase Subunit-1 (RRM1)

“…In accordance with previous published data, we confirmed that a correlation exists between RRM1 levels and resistance to gemcitabine (40,44). Gemcitabine-induced apoptosis was increased and proliferation reduced upon depletion of RRM1 with specific siRNA, confirming its key role in gemcitabine resistance.…”

“…Downregulation of RRM1 protein did not occur in response to an EGFR inhibitor (gefitinib) but was observed in MEK1/2 knocked down cells, indicating that this effect is specifically dependent on MEK inhibition. A recent study has shown that RRM1 is polyubiquitinated and identified RNF2 and Bmi1 E3 ubiquitin ligases to be involved in RRM1 degradation by the proteasome (44). Our study showed enhancement of RRM1 polyubiquitination through Lys-48-mediated linkage upon pimasertib treatment.…”

Functional Genomic Screen in Mesothelioma Reveals that Loss of Function of BRCA1-Associated Protein 1 Induces Chemoresistance to Ribonucleotide Reductase Inhibition

“…5A). In addition, we observed no clear differential pattern in RRM1 expression upon treatment, most likely due to the induction of ubiquitination and degradation of RRM1 upon gemcitabine treatment (31). However, expression of RRM2 was significantly more upregulated upon gemcitabine and hydroxyurea treatment in BAP1 mut/del compared with BAP1 WT group.…”

Lung cancer: Biology and treatment options

“…Gemcitabine directly binds to RRM1 and irreversibly inactivates ribonucleotide reductase [20–28]. High RRM1 levels are associated with tumor resistance and low RRM1 levels with tumor sensitivity to gemcitabine treatment [21,23,25–28].…”