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A mutation of Keap1 found in breast cancer impairs its ability to repress Nrf2 activity
1Schering-Plough Research Institute, 181 Passaic Avenue, Summit, NJ 07901, USA. paul.nioi@spcorp.com
Abstract:
Keap1 is the substrate recognition module of a Cullin 3-based E3 ubiquitin ligase. Its primary role is to catalyze the ubiquitylation of the Nrf2 transcription factor. Oxidative stress blocks the E3 ligase activity of Keap1 which stabilizes Nrf2 allowing it to drive the expression of certain antioxidant and drug metabolizing enzymes. A recent study identified a mutation in the Keap1 gene (Keap1C23Y) that is present in breast cancer. Using reporter gene assays we show that Keap1C23Y is impaired in its ability to repress Nrf2 dependent transcription. Unlike wild-type Keap1, we found that Keap1C23Y failed to stimulate the degradation of Nrf2. Co-immunopreciptation experiments showed that Keap1C23Y retains its ability to interact with Nrf2 and Cullin 3. In contrast, we found that Keap1C23Y could not efficiently promote the ubiquitylation of Nrf2, suggesting that its intrinsic biological activity might have been compromised. These results revealed an unexpected role for the N-terminal region of Keap1 in regulating its E3 ligase activity. Importantly, our findings suggest that a paradox exists whereby Nrf2 activity is beneficial in non-malignant cells but in cancer cells it may provide a selective advantage for clonal expansion.
Insights
A Keap1 mutation (Keap1C23Y) found in breast cancer impairs its ability to degrade Nrf2. This suggests Nrf2 activation may aid cancer cell growth, presenting a therapeutic paradox.
Area of Science:
- Molecular Biology
- Cancer Biology
- Biochemistry
Background:
- Keap1 functions as a substrate recognition module for a Cullin 3-based E3 ubiquitin ligase.
- Keap1 targets the Nrf2 transcription factor for ubiquitylation and degradation.
- Oxidative stress inhibits Keap1's E3 ligase activity, stabilizing Nrf2 and promoting antioxidant gene expression.
Purpose of the Study:
- To investigate the functional consequences of a Keap1 mutation (Keap1C23Y) identified in breast cancer.
- To determine if Keap1C23Y affects Nrf2 ubiquitylation and degradation.
- To explore the role of the N-terminal region of Keap1 in regulating E3 ligase activity.
Main Methods:
- Reporter gene assays to assess Nrf2-dependent transcription.
- Co-immunoprecipitation to analyze protein-protein interactions (Keap1, Nrf2, Cullin 3).
- Assessment of Nrf2 ubiquitylation and degradation.
Main Results:
- Keap1C23Y exhibited impaired repression of Nrf2-dependent transcription.
- Keap1C23Y failed to stimulate Nrf2 degradation, unlike wild-type Keap1.
- Keap1C23Y maintained interactions with Nrf2 and Cullin 3 but showed reduced Nrf2 ubiquitylation.
- These findings highlight an essential role for Keap1's N-terminal region in its E3 ligase function.
Conclusions:
- The Keap1C23Y mutation compromises E3 ligase activity, leading to Nrf2 stabilization.
- Nrf2 activation, while beneficial in normal cells, may confer a selective advantage for cancer cell clonal expansion.
- This presents a paradox for therapeutic strategies targeting the Keap1-Nrf2 pathway in cancer.
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