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Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter
Published on: March 27, 2020
Integrative High-Throughput RNAi Screening Identifies BRSK1, STK32C and STK40 as Novel Activators of YAP/TAZ
Mandeep K Gill1, Siyuan Song1, Tania Christova1
1Department of Biochemistry, Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada.
Abstract:
Disruption of the Hippo pathway leads to activation of the YAP/TAZ transcriptional program which promotes tumor initiation, progression and metastasis in diverse cancers. Aggressive triple-negative breast cancers (TNBC) lack an effective therapy; thus, inactivating YAP and TAZ has emerged as an attractive approach and a new treatment modality. Thus, we performed two complementary high-throughput RNAi-based kinome screens to uncover cancer-associated activators of YAP/TAZ in two TNBC cell lines, MDA-MB231 and MDA-MB468. Integrated analysis that combined a YAP/TAZ localization screen with a TEAD-luciferase reporter screen, identified novel regulators including BRSK1, STK32C and STK40. The AMPK family members NUAKs, MARKs and SIKs are known to inhibit the Hippo kinase cassette; here, we uncover BRSK1, another AMPK family member as a regulator of YAP/TAZ. We also reveal that two poorly studied kinases, STK32C, a member of the AGC family, and STK40, a pseudokinase, can also inhibit the activity of YAP/TAZ. Thus, our studies expand the repertoire of known AMPK family members and reveal two new kinases that modulate the Hippo pathway and may play a role in YAP/TAZ driven breast cancers. Further analysis of other screen hits may similarly uncover new regulators that could be targeted for therapeutic interventions.
Insights
Researchers identified new regulators of YAP/TAZ in triple-negative breast cancer (TNBC) by using high-throughput screens. These findings may lead to novel therapeutic strategies for TNBC by targeting the Hippo pathway.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Research
Background:
- The Hippo pathway regulates organ size and is frequently disrupted in cancer.
- YAP/TAZ transcriptional programs, activated by Hippo pathway disruption, drive tumor initiation, progression, and metastasis.
- Triple-negative breast cancer (TNBC) lacks effective therapies, making YAP/TAZ inhibition an attractive therapeutic strategy.
Purpose of the Study:
- To identify novel cancer-associated activators of YAP/TAZ in TNBC using high-throughput RNAi-based kinome screens.
- To uncover potential therapeutic targets for TNBC by understanding YAP/TAZ regulation.
Main Methods:
- Conducted two complementary high-throughput RNAi-based kinome screens in MDA-MB231 and MDA-MB468 TNBC cell lines.
- Integrated analysis of a YAP/TAZ localization screen with a TEAD-luciferase reporter screen.
- Utilized bioinformatics to identify and validate novel kinase regulators.
Main Results:
- Identified BRSK1, STK32C, and STK40 as novel regulators of YAP/TAZ activity in TNBC.
- Uncovered BRSK1 as a novel AMPK family member regulating YAP/TAZ.
- Revealed STK32C (AGC family kinase) and STK40 (pseudokinase) as inhibitors of YAP/TAZ activity.
Conclusions:
- Expanded the repertoire of known AMPK family members involved in Hippo pathway regulation.
- Identified STK32C and STK40 as new kinases that modulate the Hippo pathway.
- These novel regulators may play a role in YAP/TAZ-driven breast cancers and represent potential therapeutic targets.
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