The Hippo signalling pathway plays important roles in animal development, physiology and tumorigenesis1,2,3. Understanding how the activity of this pathway is regulated by the cellular microenvironment remains a major challenge. Here we elucidate a molecular mechanism by which hypoxia deactivates Hippo signalling. We demonstrate that the E3 ubiquitin ligase SIAH2 stimulates YAP by destabilizing LATS2, a critical component of the Hippo pathway, in response to hypoxia. Loss of SIAH2 suppresses tumorigenesis in a LATS2-dependent manner in a xenograft mouse model. We further show that YAP complexes with HIF1α and is essential for HIF1α stability and function in tumours in vivo. LATS2 is downregulated in human breast tumours and negatively correlates with SIAH2 expression levels, indicating that the SIAH2–LATS2 pathway may have a role in human cancer. Our data uncover oxygen availability as a microenvironment signal for the Hippo pathway and have implications for understanding the regulation of Hippo signalling in tumorigenesis.
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We thank H. Deng from Tsinghua University for Mass spectrometry analysis, Y. (YZ) Luo from Tsinghua University for providing the HRE–luciferase reporter plasmid and Y. (YG) Chen from Tsinghua University for providing the HA–VHL plasmid. We are deeply grateful to D. (DJ) Pan from Johns Hopkins University for his suggestions during the manuscript revision. This work was supported by the National Basic Research Program of China (2010CB912204, 2011CB9109903, 2011CB943903 and 2013CB531200) and the National Natural Sciences Foundation of China (31471319, 81130045 and 31171411).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 5 Schematic drawing of LATS2 deletion mutants and their responses to interaction with and degradation by SIAH2.
A schematic diagram of LATS2 deletion mutants is shown on the left panel. Blue represents as LATS Conserved Domain 1(LCD1) (aa. 1–160), yellow as LATS Conserved Domain 2(LCD2) (aa. 403–463), red as PAPA repeat (aa. 467–480), green as Protein Binding Domain (PBD) (aa. 618–720), purple as Kinase Domain. LATS2 binding activity to SIAH2 is shown in the middle panel as assayed in Fig. 1d. “+” indicates as binding, “−” as no binding, and “−/+” as less binding. The right panel shows the responses of LATS2 mutants to SIAH2-induced degradation. Cells expressing constant amounts of Myc-LATS2 mutant and increasing amounts of Flag-SIAH2 were subjected to immunoblotting. “+” indicates response to SIAH2-induced degradation and “−” indicates non-response.
(a,b) LATS2 degradation was dosage-dependent on SIAH2 (a), but not on SIAH2RM, an E3 ligase-activity-dead mutant of SIAH2 (b). (c,d) SIAH2-mediated LATS2 degradation was inhibited by proteasomal inhibitor MG132 (c), but not by lysosomal inhibitor BA-1 (d). (e,f) The half-life of LATS2 was shortened by SIAH2 (e) but not by SIAH2RM (f) in cycloheximide chase assays.
(a) No co-immunoprecipitation of exogenously expressed Myc-LATS2 by Flag-SIAH1RM. (b) LATS2 degradation is specifically promoted by SIAH2 but not by SIAH1. (c) Co-immunoprecipitation of exogenous expressed Flag-SIAH2 by Myc-LATS1 and vice versa. (d) LATS1 stability is regulated by SIAH2 but not by SIAH2RM. (e) No co-immunoprecipitation of exogenously expressed Myc-LATS1 by Flag-SIAH1RM.
(a) Scramble and YAP knockdown MDA-MB-231 cells were cultured under hypoxic conditions. Total RNA was extracted and subjected to RT-qPCR analysis for HIF1α mRNA expression. (b) MG132 stabilized HIF1α in YAP knockdown cells under hypoxic conditions. (c) Scramble and YAP knockdown MDA-MB-231 cells were cultured under hypoxic conditions in the presence of MG132. Protein expressions were analysed by immunoblotting with indicated antibodies. Data in a is the mean of n = 3 independent experiments and error bars indicate s.d. Two-tailed, unpaired Student’s t-test.
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Ma, B., Chen, Y., Chen, L. et al. Hypoxia regulates Hippo signalling through the SIAH2 ubiquitin E3 ligase. Nat Cell Biol 17, 95–103 (2015). https://doi.org/10.1038/ncb3073
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