A RAS-CaMKKβ-AMPKα2 pathway promotes senescence by licensing post-translational activation of C/EBPβ through a novel 3′UTR mechanism

Abstract

Oncogene-induced senescence (OIS) is an intrinsic tumor suppression mechanism that requires the p53 and RB pathways and post-translational activation of C/EBPβ through the RAS-ERK cascade. We previously reported that in transformed/proliferating cells, C/EBPβ activation is inhibited by G/U-rich elements (GREs) in its 3′UTR. This mechanism, termed “3′UTR regulation of protein activity” (UPA), maintains C/EBPβ in a low-activity state in tumor cells and thus facilitates senescence bypass. Here we show that C/EBPβ UPA is overridden by AMPK signaling. AMPK activators decrease cytoplasmic levels of the GRE binding protein HuR, which is a key UPA component. Reduced cytoplasmic HuR disrupts 3′UTR-mediated trafficking of Cebpb transcripts to the peripheral cytoplasm – a fundamental feature of UPA – thereby stimulating C/EBPβ activation and growth arrest. In primary cells, oncogenic RAS triggers a Ca++-CaMKKβ-AMPKα2-HuR pathway, independent of AMPKα1, that is essential for C/EBPβ activation and OIS. This axis is disrupted in cancer cells through down-regulation of AMPKα2 and CaMKKβ. Thus, CaMKKβ-AMPKα2 signaling constitutes a key tumor suppressor pathway that activates a novel UPA-cancelling mechanism to unmask the cytostatic and pro-senescence functions of C/EBPβ.

3’UTR-directed, kinase proximal mRNA decay inhibits C/EBPβ phosphorylation/activation to suppress senescence in tumor cells

“…RNA FISH labeling showed exclusion of Cebpb mRNAs from the perinuclear region (Fig. S2A), as observed previously (Basu et al, 2018a). Interestingly, UPF1 was restricted to the perinuclear area that was largely devoid of Cebpb FISH signals.…”

“…To begin to address whether the GRE Δ/Δ genotype confers resistance to RAS-induced transformation, we crossed the mutant strain to mice lacking the p19 Arf tumor suppressor and prepared MEFs of four genotypes: WT , GRE Δ/Δ , p19 Arf−/− , and p19 Arf−/− ;GRE Δ/Δ . As C/EBPβ UPA is abrogated in RIS cells but not in RAS-transformed tumor cells (Basu et al, 2018a) and p19 Arf deficiency circumvents senescence to facilitate neoplastic transformation, we reasoned that ΔGRE phenotypes may be more apparent in cells lacking p19 Arf . RAS-induced phosphorylation on Ser222 was enhanced more ∼1.7-fold in p19 Arf−/− ;GRE Δ/Δ MEFs compared to p19 Arf−/− cells (Fig.…”

“…UPA is abrogated in primary cells undergoing oncogenic RAS-induced senescence (RIS), allowing C/EBPβ to become activated by senescence stimuli. UPA is disabled by a RAS- CaMKKβ-AMPK pathway that induces nuclear translocation of HuR/ELAVL1 (Basu et al, 2018a), an RNA-binding protein that recognizes a G/U-rich element (GRE) region in the Cebpb 3’UTR (Basu et al, 2011; Gantt et al, 2005). HuR and the GRE motif are both required for C/EBPβ UPA, as HuR ablation or deletion of the GRE abrogate 3’UTR inhibition and disrupt peripheral Cebpb mRNA localization (Basu et al, 2011).…”

The “Janus” Role of C/EBPs Family Members in Cancer Progression

“…Thus, the intracellular site of the C/EBPβ translation is critical for RAS-induced activation via effector kinases such as ERK. Interestingly, 3′UTR inhibition and C/EBPβ mRNA compartmentalization are not observed in primary mouse and human fibroblasts [ 64 ].…”

The RNA-Binding Protein HuR in Digestive System Tumors

“…Interestingly, cumulative studies have shown that HuR promotes cell senescence, which is closely related to tumors, by increasing reactive oxygen species (ROS) by enhancing the mitochondrial localization of the telomeric protein TIN2 and increasing Foxp3 expression, activating oncogene-induced senescence via the Ca ++ -CaMKK β -AMPK α 2-HuR pathway, etc. [ 27 – 29 ]. Therefore, studying the functional properties of HuR plays a crucial role in the future prevention and treatment of tumors.…”