Dual degradation signals destruct GLI1: AMPK inhibits GLI1 through β-TrCP-mediated proteasome degradation

Abstract

Overexpression of the GLI1 gene has frequently been found in various cancer types, particularly in brain tumors, in which aberrant GLI1 induction promotes cancer cell growth. Therefore, identifying the molecular players controlling GLI1 expression is of clinical importance. Previously, we reported that AMPK directly phosphorylated and destabilized GLI1, resulting in the suppression of the Hedgehog signaling pathway. The current study not only demonstrates that AMPK inhibits GLI1 nuclear localization, but further reveals that β-TrCP plays an essential role in AMPK-induced GLI1 degradation. We found that activation of AMPK promotes the interaction between β-TrCP and GLI1, and induces β-TrCP-mediated GLI1-ubiquitination and degradation. Inhibiting AMPK activity results in the dissociation of the β-TrCP and GLI1 interaction, and diminishes β-TrCP-mediated-GLI1 ubiquitination and degradation. On GLI1, substitution of AMPK phosphorylation sites to aspartic acid (GLI13E) results in stronger binding affinity of GLI1 with β-TrCP, accompanied by enhanced GLI1 ubiquitination and later degradation. In contrast, the GLI1 alanine mutant (GLI13A) shows weaker binding with β-TrCP, which is accompanied by reduced β-TrCP-mediated ubiquitination and degradation. Together, these results demonstrate that AMPK regulates GLI1 interaction with β-TrCP by phosphorylating GLI1 and thus both post-translational modifications by AMPK and β-TrCP ultimately impact GLI1 degradation.

Loss of AMPKα2 Impairs Hedgehog-Driven Medulloblastoma Tumorigenesis

“…The apparent discrepancy between our results observed in mouse and the previously described tumor-suppressive role of AMPK in human medulloblastoma cell lines [ 28 , 29 , 30 , 33 ] could be due to the species-specific role of AMPK in the cerebellum. While all studies in which the role of AMPK has been directly tested genetically in human medulloblastoma cells agree, i.e., AMPK suppresses SHH-driven cell growth, the role of AMPK in mouse cell lines is debated [ 28 , 29 , 30 , 33 ]. For example, several studies by the Yang laboratory showed that AMPK phosphorylates GLI1 at multiple sites and targets it for proteasomal degradation in murine cells [ 28 , 29 , 33 ].…”

“…Here, we describe the analysis of loss of AMPKα2 in a genetically engineered mouse model of SHH-driven medulloblastoma. Remarkably, in disagreement with the previous studies supporting a tumor-suppressive role for AMPK in medulloblastoma tumorigenesis [ 28 , 29 , 30 , 31 , 32 , 33 ], our analysis reveals that loss of AMPKα2 impairs SHH-driven medulloblastoma tumorigenesis.…”

“…In this study, we demonstrate that loss of AMPKα2 impairs SHH-driven medulloblastoma tumorigenesis. This is a surprising observation given the previous reports supporting a tumor-suppressive role for AMPK in medulloblastoma tumorigenesis [ 28 , 29 , 30 , 31 , 32 , 33 ] and the fact that the subunit AMPKα2 alone can suppress tumorigenesis in other contexts [ 21 , 45 , 46 ]. In fact, together with a previous analysis of AMPKα2 in mammary carcinoma [ 25 ], our analysis represents the first in vivo evidence that the loss of AMPKα2 alone can impair oncogenesis.…”

“…On the other hand, maintenance of the metabolic balance by AMPK may be critical for survival during metabolic stress that can occur in the tumor microenvironment—hence the requirement for AMPK activity in some cancer cells [ 22 , 23 , 24 , 25 , 26 , 27 ]. Similarly, in medulloblastoma, the role of AMPK as both an inhibitor of GLI1 and SHH signaling [ 28 , 29 , 30 , 33 ] (tumor-suppressive effect) as well as a promoter of CNBP and polyamine metabolism (pro-tumorigenic effect) supposes a dual role for AMPK in this disease context. In agreement with the observation that CNBP protein levels are decreased in the absence of AMPKα2, we observed that the pro-tumorigenic function of AMPKα2 prevails in the context of the highly penetrant [GFAP-tTA;TRE-SMOA1] mouse model of medulloblastoma.…”

“…In SHH-driven medulloblastoma tumorigenesis, several studies support a tumor-suppressive role for AMPK [ 28 , 29 , 30 , 31 , 32 , 33 ]. First, in medulloblastoma cells, AMPK phosphorylates the SHH pathway transcription factor GLI1, promoting its proteasomal degradation, thus inhibiting SHH signaling and SHH-driven medulloblastoma [ 28 , 29 , 30 , 33 ]. Second, a potential tumor-suppressive role for AMPK in SHH-driven medulloblastoma was also inferred by the fact that the increased survival observed upon Hk2 KO in a mouse model of medulloblastoma is accompanied by a gain of AMPK activity [ 31 , 32 ].…”