GSK3α Regulates Temporally Dynamic Changes in Ribosomal Proteins upon Amino Acid Starvation in Cancer Cells

Abstract

Amino acid availability is crucial for cancer cells’ survivability. Leukemia and colorectal cancer cells have been shown to resist asparagine depletion by utilizing GSK3-dependent proteasomal degradation, termed the Wnt-dependent stabilization of proteins (Wnt/STOP), to replenish their amino acid pool. The inhibition of GSK3α halts the sourcing of amino acids, which subsequently leads to cancer cell vulnerability toward asparaginase therapy. However, resistance toward GSK3α-mediated protein breakdown can occur, whose underlying mechanism is poorly understood. Here, we set out to define the mechanisms driving dependence toward this degradation machinery upon asparagine starvation in cancer cells. We show the independence of known stress response pathways including the integrated stress response mediated with GCN2. Additionally, we demonstrate the independence of changes in cell cycle progression and expression levels of the asparagine-synthesizing enzyme ASNS. Instead, RNA sequencing revealed that GSK3α inhibition and asparagine starvation leads to the temporally dynamic downregulation of distinct ribosomal proteins, which have been shown to display anti-proliferative functions. Using a CRISPR/Cas9 viability screen, we demonstrate that the downregulation of these specific ribosomal proteins can rescue cell death upon GSK3α inhibition and asparagine starvation. Thus, our findings suggest the vital role of the previously unrecognized regulation of ribosomal proteins in bridging GSK3α activity and tolerance of asparagine starvation.

GSK3 as a Master Regulator of Cellular Processes

“…Subsequent RNA-sequencing analyses revealed that GSK3α KD cells surviving asparagine depletion were characterized by a downregulation of various ribosomal proteins (RP). Moreover, the suppression of specific RP in GSK3α KO cells was sufficient to protect them from asparaginase-induced cell death, collectively suggesting a detrimental influence of these RP on GSK3α-dependent cancer cell survival under these conditions [24]. In the next study, metabolic regulation was revisited as one of the most prominent functions of GSK3.…”

“…The next three studies presented in this Special Issue are dedicated to the impact of GSK3 modulation on central cellular processes, i.e., cell cycle progression [23], the management of amino acid (aa) deprivation [24], and metabolism [25]. Shenker et al, for instance, were interested in elucidating the cytotoxic properties of Aggregatibacter actinomycetemcomitans-derived cytolethal distending toxin (AaCdt) [23], a heterotrimeric bacterial toxin possessing both DNase and lipid phosphatase activity, which typically causes cell cycle arrest and apoptosis [26].…”

“…Correspondingly, AaCdt-induced G2/M arrest decreased in the presence of GSK3 inhibitors, indicating that the AaCdtmediated inhibition of cell cycle progression involves GSK3β enzymatic activity [23]. In another paper, Loxha et al had a closer look at GSK3α-regulated cancer cell survival under aa deprivation [24], a mechanism contributing to the resistance of cancer cells towards aa depletion (as practiced, for instance, by asparaginase application) by releasing proteinbound aa via enhanced GSK3α-driven protein degradation [28]. Hence, GSK3α knockdown (KD) in T-cell acute lymphoblastic leukemia and colorectal cancer cells massively reduced cell viability and protein ubiquitination in the presence of arginase, while cell death significantly increased [24].…”

“…In another paper, Loxha et al had a closer look at GSK3α-regulated cancer cell survival under aa deprivation [24], a mechanism contributing to the resistance of cancer cells towards aa depletion (as practiced, for instance, by asparaginase application) by releasing proteinbound aa via enhanced GSK3α-driven protein degradation [28]. Hence, GSK3α knockdown (KD) in T-cell acute lymphoblastic leukemia and colorectal cancer cells massively reduced cell viability and protein ubiquitination in the presence of arginase, while cell death significantly increased [24]. In the latter, however, the pro-apoptotic aa deficiencyinduced GCN2 ("general control nonderepressible 2")-eukaryotic translation initiation factor (eIF) 2α-activating transcription factor (ATF) 4-C/EBP Homologous Protein (CHOP) axis [29] and cell cycle alterations were not involved.…”