This growth inhibition effect is not limited to these agents, as the AKT inhibitor AZD5363 and PIM inhibitor AZD1208 also abrogated proliferation of these prostate cancer cells (Supplementary Fig. Synergistic inhibition of survival and growth was demonstrated when PC3-LN4 cells were treated with both PIM and PI3K/AKT inhibitors, PIM447 and buparlisib, respectively (Supplementary Fig. S1A), and are relatively resistant to buparlisib ( Fig. PC3-LN4 cells are a highly metastatic variant of PC-3 cells ( 29, 30) that have increased PIM transcript levels compared with other prostate cancer cell lines (Supplementary Fig.
In PIM1-overexpressing LNCaP prostate cancer cells (which contain a highly activated AKT pathway secondary to deletion of PTEN), simultaneously inhibiting both PI3K and the PIM kinase enhanced growth inhibition ( Fig.
However, when PIM1 is overexpressed, these cells become highly resistant to this inhibitor ( Fig. Proliferation of human prostate cancer cells, e.g., LNCaP and PC-3 lines that express constitutively active AKT, can be inhibited by the pan-PI3K inhibitor buparlisib. Immunoblotting, immunofluorescence, and IHC A detailed description is provided in the Supplementary Methods. Alt-R CRISPR-Cas9 System is used for genome editing of the eIF4B serine 406 site. Lentiviruses were produced in 293T cells coexpressing the packaging vectors (psPAX2 and VSV-G), concentrated by ultracentrifugation (20,000 g for 2 hours at 4☌), and resuspended with culture media for an infection (48 hours). eIF4B sgRNA CRISPR-Cas9 system was purchased from ABM Inc. Cells were transfected with RNAi or transduced with lentiviruses as previously described ( 23, 26).
The cell lines were routinely tested for mycoplasma and used for fewer than 50 passages. All cell lines were maintained at 37☌ in 5% CO 2 and were authenticated by short tandem repeat DNA profiling performed by the University of Arizona Genetics Core Facility. Mouse prostate epithelial cells were grown in DMEM medium supplemented with 2.5% charcoal-stripped FCS, 5 μg/mL of insulin/transferring/selenium, 10 μg/mL of bovine pituitary extract, 10 μg/mL of epidermal growth factor, and 1 μg/mL of cholera toxin as described previously ( 25). The human prostate cancer cell lines LNCaP, PC-3, and PC3-LN4 were cultured as previously described ( 23, 24). Thus, therapeutic agents targeting NRF2 signaling may render cellular vulnerability to PI3K-AKT–inhibitory drugs. The activated NRF2 promotes the further activation of the PI3K-AKT pathway to accelerate aggressive cancer progression. Recently, it was reported that NRF2 promotes EGFR autocrine signaling to facilitate eIF4F complex formation for cap-dependent translation initiation in KRAS-mutant pancreatic adenocarcinomas ( 13). The activated NRF2, in addition to cytoprotective function, also promotes metabolic reprogramming by activating metabolic genes ( 12). AKT-stimulated mitogenic and survival programs ( 10) control ROS levels through nuclear factor erythroid 2-related factor 2 (NRF2), a basic leucine zipper transcription factor, that binds to antioxidant response elements (ARE) to activate a battery of cytoprotective and antioxidant gene targets including heme oxygenase (HMOX1), NAD(P)H quinone dehydrogenase (NQO1), glutamate-cysteine ligases, glutathione peroxidases (GPX), and superoxide dismutases (SOD ref. Because excessive oxidative stress can lead to senescence or cell death, proliferating cancer cells limit cellular ROS levels by inducing an array of antioxidants and ROS scavengers ( 9). Treatment with PIM kinase inhibitors reverses this resistance phenotype, making tumors increasingly susceptible to small-molecule therapeutics, which block the PI3K-AKT pathway.Īctivation of the AKT pathway produces reactive oxygen species (ROS). Importantly, PIM also controls NAD(P)H production by increasing glucose flux through the pentose phosphate shunt decreasing ROS production, and thereby diminishing the cytotoxicity of PI3K-AKT inhibitors. PIM prevents cell death induced by PI3K-AKT–inhibitory drugs through a noncanonical mechanism of NRF2 ubiquitination and degradation and translational control of NRF2 protein levels through modulation of eIF4B and mTORC1 activity. PIM1 kinase functions to decrease cellular ROS levels by enhancing nuclear factor erythroid 2-related factor 2 (NRF2)/antioxidant response element activity.
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Our study unveils the pivotal control of redox signaling by PIM kinases as a driver of this resistance mechanism. However, the exact mechanism by which PIM kinase promotes tumor cell resistance is unknown. Cancer resistance to PI3K inhibitor therapy can be in part mediated by increases in the PIM1 kinase.
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