The APC/C E3 Ligase Complex Activator FZR1 Restricts BRAF Oncogenic Function

Abstract

BRAF drives tumorigenesis by coordinating the activation of RAS/RAF/MEK/ERK oncogenic signaling cascade. However, upstream pathway(s) governing BRAF kinase activity and protein stability remains undefined. Here, we report that in primary cells with active APCFZR1, APCFZR1 earmarks BRAF for ubiquitination-mediated proteolysis, while in cancer cells with APC-free FZR1, FZR1 suppresses BRAF through disrupting BRAF dimerization. Moreover, we identified FZR1 as a direct target of ERK and CYCLIN D1/CDK4 kinases. Phosphorylation of FZR1 inhibits APCFZR1, leading to elevation of a cohort of oncogenic APCFZR1 substrates to facilitate melanomagenesis. Importantly, CDK4 and/or BRAF/MEK inhibitors restore APCFZR1 E3 ligase activity, which might be critical for their clinical effects. Furthermore, FZR1 depletion co-operates with AKT hyper-activation to transform primary melanocytes, while genetic ablation of Fzr1 synergizes with Pten loss, leading to aberrant co-activation of BRAF/ERK and AKT signaling in mice. Our findings therefore reveal a reciprocal suppression mechanism between FZR1 and BRAF in controlling tumorigenesis.

Cyclin D–CDK4 kinase destabilizes PD-L1 via cullin 3–SPOP to control cancer immune surveillance

“…The construct of HA-PD-L1 (HA tag in the N-terminus of PD-L1) was kindly provided by Dr. Mien-Chie Hung (The University of Texas MD Anderson Cancer Center). HA-Cdh1, HA-Cdc20, shCdh1, hCdc20 and HA-14-3-3 isoform constructs were described previously 33, 34 . pCMV-CDK4 WT, pCMV-CDK4 N158F and shcyclin D3 were described previously 35, 36 .…”

Ubiquitin ligases in oncogenic transformation and cancer therapy

“…Interestingly, ERK and CDK4 both phosphorylate CDH1, which decreases its association with APC/C and leads to the accumulation of APC/C substrates, representing a positive feedback loop between hyperactivated ERK signalling and BRAF stability, as demonstrated in multiple melanoma cell lines and in BRAF-V600E-expressing immortalized mouse melanocytes. Consistent with this, treatment of cells with ERK inhibitors or CDK inhibitors decreases BRAF protein levels, suggesting that combined inhibition of these kinases is a useful therapy for cancers with hyperactivated BRAF 83 . Importantly, CDH1 can also limit signalling in cancer cells expressing WT BRAF (or dimerization-dependent BRAF mutants); in this case, direct binding of CDH1 prevents BRAF dimerization and full activation 83 .…”

“…Consistent with this, treatment of cells with ERK inhibitors or CDK inhibitors decreases BRAF protein levels, suggesting that combined inhibition of these kinases is a useful therapy for cancers with hyperactivated BRAF 83 . Importantly, CDH1 can also limit signalling in cancer cells expressing WT BRAF (or dimerization-dependent BRAF mutants); in this case, direct binding of CDH1 prevents BRAF dimerization and full activation 83 . The dual-suppressor activity of CDH1 on BRAF illustrates the need to develop CDC20-specific inhibitors 21,83 .…”

“…When HSP90 activity is inhibited pharmacologically, its client proteins, including BRAF-V600E, are degraded by cullin-5-mediated ubiquitylation and proteasomal degradation 81,82 . CDH1 has also been suggested to limit BRAF signalling by multiple mechanisms 83 . In non-malignant cells, the APC/C–CDH1 complex induces BRAF proteolysis in a cell-cycle-dependent manner, but this process is suppressed in BRAF-V600E-expressing melanoma cells 83 .…”

“…CDH1 has also been suggested to limit BRAF signalling by multiple mechanisms 83 . In non-malignant cells, the APC/C–CDH1 complex induces BRAF proteolysis in a cell-cycle-dependent manner, but this process is suppressed in BRAF-V600E-expressing melanoma cells 83 . Interestingly, ERK and CDK4 both phosphorylate CDH1, which decreases its association with APC/C and leads to the accumulation of APC/C substrates, representing a positive feedback loop between hyperactivated ERK signalling and BRAF stability, as demonstrated in multiple melanoma cell lines and in BRAF-V600E-expressing immortalized mouse melanocytes.…”