Abstract
Cancer resistance to PI3K inhibitor therapy can be in part mediated by increases in the PIM1 kinase. However, the exact mechanism by which PIM kinase promotes tumor cell resistance is unknown. Our study unveils the pivotal control of redox signaling by PIM kinases as a driver of this resistance mechanism. PIM1 kinase functions to decrease cellular ROS levels by enhancing nuclear factor erythroid 2-related factor 2 (NRF2)/antioxidant response element activity. 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. 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. Treatment with PIM kinase inhibitors reverses this resistance phenotype, making tumors increasingly susceptible to small-molecule therapeutics, which block the PI3K-AKT pathway.
Introduction
Intrinsic and acquired resistance to PI3K inhibitors has been poorly understood, and recent studies in breast cancer identified the PIM (Proviral Integration site for Moloney murine leukemic virus) protein kinase as additional therapeutic targets that sensitize cancer cells to anticancer therapeutics that inhibit this pathway (1). These findings are highly relevant to prostate cancer where PIM is highly expressed and aberrations in the PI3K pathway and deletion of PTEN are detected in nearly 70% of patients with metastatic castration-resistant prostate cancer (mCRPC; ref. 2). The prevalence of PI3K pathway activation in mCRPC cells makes this pathway an attractive target to inhibit tumor growth. However, in clinical trials PI3K inhibitor therapy induced a partial response in only 12% of patients whose tumors harbor mutations in PTEN or PIK3C isoforms (3), suggesting a possible role for PIM kinase in blocking this pathway.
PIM kinases, a family comprised of PIM1, PIM2, and PIM3, are oncogenic serine/threonine kinases that are overexpressed in multiple malignancies (4, 5) that promote tumor progression by regulating cell-cycle progression, proliferation, and survival (6). PIM1 expression is elevated in approximately 50% of human prostate cancer specimens, particularly in high Gleason grade and aggressive metastatic prostate cancer cases (5, 7). These protein kinases share significant mechanistic similarities to the AKT family which might account for their ability to act as part of a resistance mechanism. For example, both PIM and AKT families are known to regulate protein translation by affecting activity of mTORC1, a nutrient/energy/redox sensor and regulator of protein synthesis, through an ability to modify identical proteins. PIM and AKT overlap in substrate modification; PIM phosphorylates TSC2, PRAS40, and eIF4B on serine (S) 406 (8).
Activation of the AKT pathway produces reactive oxygen species (ROS). 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). 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 [GCLC and GCLM; the first rate-limiting enzymes of glutathione (GSH) synthesis], glutathione peroxidases (GPX), and superoxide dismutases (SOD; ref. 11). The activated NRF2, in addition to cytoprotective function, also promotes metabolic reprogramming by activating metabolic genes (12). 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 promotes the further activation of the PI3K-AKT pathway to accelerate aggressive cancer progression. Thus, therapeutic agents targeting NRF2 signaling may render cellular vulnerability to PI3K-AKT–inhibitory drugs.
Previously, we reported that PIM kinase mediates an important adaptive survival response upon inhibition of AKT through feedback activation of receptor tyrosine kinase signaling, leading to cancer cell resistance (14). Recently, a genome scale shRNA screening identified the PIM kinase as conferring resistance to the PI3K inhibitor alpelisib (BYL719; ref. 1). Concurrent treatment with a PIM inhibitor and PI3K inhibitor overcame this resistance. Moreover, PIM1 was identified as a pivotal therapeutic target in triple-negative breast cancer (15, 16), inhibiting PIM sensitized these cells to chemotherapy.
In this study, we have defined the potential mechanism underlying the PIM1-mediated inhibition of PI3K-AKT inhibitor in both tissue culture and mouse models harboring PI3K-AKT pathway aberrations.
Materials and Methods
Chemicals
Cell culture, stable cell lines, transfection, and CRISPR-Cas9
The human prostate cancer cell lines LNCaP, PC-3, and PC3-LN4 were cultured as previously described (23, 24). 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). All cell lines were maintained at 37°C in 5% CO2 and were authenticated by short tandem repeat DNA profiling performed by the University of Arizona Genetics Core Facility. The cell lines were routinely tested for mycoplasma and used for fewer than 50 passages. Cells were transfected with RNAi or transduced with lentiviruses as previously described (23, 26). eIF4B sgRNA CRISPR-Cas9 system was purchased from ABM Inc. 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°C), and resuspended with culture media for an infection (48 hours). Alt-R CRISPR-Cas9 System is used for genome editing of the eIF4B serine 406 site. A detailed description is provided in the Supplementary Methods.
Immunoblotting, immunofluorescence, and IHC
For immunoblotting, total lysates were prepared, resolved by SDS-PAGE, and transferred to immobilon membranes. The following primary antibodies were utilized from the indicated vendors: PIM1, p-IRS1 (S1101), γ-H2AX (S139), p-AKT (S473), AKT, p-p70S6K (T389), p70S6K, and pS6 (S240/244) were from Cell Signaling Technology; S6, NRF2, HMOX1, GCLC, GCLM, and PRDX3 were from Santa Cruz Biotechnology; SOD2 was from Abcam; ACTIN, FLAG, HA, and ubiquitin antibodies were purchased from Sigma. IHC and immunofluorescence staining were performed as described in detail in the Supplementary Methods. Staining with H2DCF-DA followed by flow cytometry was as previously described (26).
Metabolic analysis
Lactate dehydrogenase, NADPH, and GSH production was assessed using an Amplite Colorimetric L-Lactate Dehydrogenase (LDH) Assay Kit (AAT Bioquest), bioluminescent cell–based NAD(P)/NAD(P)H assay, and GSH-Glo kits (Promega), respectively, and was carried out as per the manufacturer's instructions. Measurement of oxygen consumption rate (OCR) and extracellular acidification was performed using a Seahorse Bioscience XF24 Extracellular Flux Analyzer. Following exposure of LNCaP to [U-13C] glucose, carbon flux through metabolic pathways was examined at the University of Michigan Regional Comprehensive Metabolomics Resource Core (http://mrc2.umich.edu).
Animals
For xenograft studies, male SCID mice (8 weeks old) were used by approval of the Institutional Animal Care and Use Committee at the University of Arizona and described in detail in the Supplementary Methods.
Prostate cancer patient-derived organoids
Prostate cancer biopsies were provided by University of Arizona Cancer Center Tissues Acquisition and Cellular/Molecular Analysis Shared Resources, and the study was conducted under University of Arizona Institutional Review Board approval. Prostate cancer patient-derived organoids were cultured based on previously established procedures (27, 28) and are described in detail in the Supplementary Methods.
Statistical analysis
Statistical significance was determined, when appropriate, using unpaired, two-tailed Student t test. P values of less than 0.05 were considered statistically significant.
Results
PIM kinase and PI3K-AKT inhibitors synergize to suppress tumor growth
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. However, when PIM1 is overexpressed, these cells become highly resistant to this inhibitor (Fig. 1A and B). 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. 1C). 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. S1A), and are relatively resistant to buparlisib (Fig. 1D and E). 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. S1B). 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. S1C–S1E). To determine whether these effects were observed in vivo, mice were xenografted with PC3-LN4 tumors and then treated with buparlisib (10 mg/kg) with or without PIM447 (30 mg/kg) for 24 days. The combination treatment significantly inhibited in vivo growth, indicated by a decrease in tumor volume and weight compared with animals treated with either buparlisib, PIM447, or vehicle control (Fig. 1F and G). The combination treatment significantly reduced tumor cell proliferation compared with either agent alone (Supplementary Fig. S1F and S1G), evidenced by the marked decrease in Ki67 staining. Similar results were obtained using the AKT inhibitor AZD5363 (40 mg/kg) in place of buparlisib (Fig. 1H and I). These results suggest that tumor cell resistance to PI3K/AKT inhibitors was mediated, at least in part, by the PIM kinases.
PIM1 kinase induces tumor cell resistance to inhibitors of PI3K/AKT signaling by increasing NRF2 levels and stimulating the production of ROS scavengers
Small-molecule PIM inhibitors inactivate the NRF2 transcription factor, reducing expression of cytoprotective genes and leading to the buildup of ROS in hypoxia (31). To understand the biochemical mechanism by which PIM kinases cause resistance to inhibitors of PI3K/AKT, the relationship between NRF2 signaling and PIM1 kinase was explored. To minimize genetic alterations affecting NRF2 regulation, we utilized mouse epithelial prostate cancer (mPrEC) cells derived from spontaneous prostate adenocarcinomas that arose in a mouse model Trp53loxP/loxP; Rb1loxP/loxP; PbCre4 (Supplementary Fig. S2A). When PIM1 was expressed in cells from these mice (mPrEC/PIM1), these cells established larger colonies in soft agar (Supplementary Fig. S2B) and had elevated levels of NRF2-ARE genes including HMOX1, NQO1, SOD2, and SOD3 (Supplementary Fig. S2C; GEO repository accession number, GSE118786). Because activated AKT was detected in mPrEC/PIM1 cells, these cells were treated with buparlisib together with PIM447. Treatment blocked AKT and PIM activity, judged by inhibition of phospho-AKT (S473) and phospho-IRS1 (S1101) expression, respectively, which resulted in inhibition of mTORC1 and NRF2 activity (Fig. 2A); the effects of combination treatment further resulted in DNA damage as measured by the marked induction of γ-H2AX expression. Sensitivity of mPrEC cells to buparlisib was markedly enhanced by cotreatment with PIM447 (Fig. 2B). These results suggest that in prostate cell lines with few mutations, combining PI3K and PIM inhibitors may be useful in blocking tumor growth. To test activity of combination therapy to kill prostate cancer from primary patient samples, the laboratory has used organoids cultured from patient biopsies of the prostate obtained from prostatectomy specimens. These patient organoids had activated AKT and increased PSA levels (Fig. 2C; Supplementary Fig. S2D and S2E). qPCR analysis confirmed the presence of PIM1, 2, and 3 transcripts in these organoids (Supplementary Fig. S2F). These organoids had detectible levels of the PIM substrate, phopho-IRS1 (S1101), indicative of PIM kinase activity (23). Organoid data resembled cell culture and animal models as combination treatment of buparlisib and PIM447 resulted in a significant reduction in organoid number and viability (Fig. 2D).
To examine whether NRF2 signaling induced by PIM1 kinase plays a role in resistance of human prostate cancer cell to PI3K inhibitor, a Doxycycline (Dox)-inducible vector was used to induce PIM1 overexpression in LNCaP cells; NRF2 protein expression was markedly increased (Supplementary Fig. S2G) without significant increase of NRF2 mRNA. This increase in NRF2 protein induced multiple downstream targets of NRF2, including ROS scavengers (HMOX1 and NQO1), an enzyme needed for GSH synthesis (GCLM), and enzymes that modulate cellular NADPH levels (i.e., ME1, IDH1, and G6PD; Supplementary Fig. S2H). To demonstrate that the increase in ROS scavengers was secondary to PIM1-mediated NRF2 induction, and not a direct effect of elevating the PIM kinase, NRF2-targeted shRNA was expressed in the human prostate cancer cells containing Dox-inducible PIM1. PIM1-mediated induction of HMOX1 and NQO1 expression was abrogated by depletion of NRF2 (Fig. 2E; Supplementary Fig. S2I). Treatment of LNCaP cells with buparlisib, a pan PI3K inhibitor, blocks AKT phosphorylation and markedly decreases NRF2 levels, leading to reduction in ROS scavengers, NQO1, HMOX1, SOD2, and in the GCLM enzyme. In contrast, when the expression of PIM1 was increased even in the presence of buparlisib treatment and blocked AKT phosphorylation, there was no change in the ROS scavenger protein levels (Fig. 2F). Over a broad range of doses, buparlisib failed to downregulate HMOX1 in PIM1-overexpressing prostate cancer cells (Supplementary Fig. S2J).
Modulation of ROS scavengers is important for buparlisib-mediated LNCaP tumor cell killing as evidenced by the fact that forced NRF2 or HMOX1 expression conferred buparlisib resistance (Fig. 2G). Conversely, depletion of NRF2 in the resistant PIM1 prostate cancer lines rendered these cells susceptible to buparlisib-induced killing (Fig. 2H). Depletion of HMOX1 expression in the PIM1-overexpressing cells also restored cell sensitivity to buparlisib, as evidenced by cleavage of PARP-1, a marker of apoptosis, and markedly increased cell death (Fig. 2I). In PC3-LN4 cells, siRNA-mediated depletion of HMOX1 markedly increased buparlisib-induced cell death as characterized by caspase-3 and PARP-1 cleavage (Fig. 2J). Therefore, PIM1 prevents small-molecule PI3K inhibitor–induced cell death by regulating the levels of NRF2 and ROS scavenger proteins.
Redox regulation of GSH is a critical factor in PIM-mediated resistance to PI3K-AKT inhibitor
Luminescence-based assays demonstrate that increased expression of PIM1 blocks the ability of buparlisib treatment to decrease the cellular levels of both NADPH and GSH (Fig. 3A). To further examine the role of ROS in the regulation of cell death induced by PI3K inhibitors, LNCaP/PIM1 were treated with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. PIM1 transduction made these cells less sensitive to BSO killing (Fig. 3B), in comparison with control cells (LNCaP-expressing empty vector, LNCaP/EV). To test whether the addition of BSO significantly suppressed in vivo tumor growth, LNCaP/PIM1 cells were grown subcutaneously in immunocompromised mice, and animals were treated with BSO (400 mg/kg), buparlisib, or the combination. Combination treatment with these agents inhibited tumor growth significantly better than either agent alone (Fig. 3C). To document ROS stimulation by these agents, prostate cells were stained with H2DCF-DA. In LNCaP/EV, buparlisib increased ROS accumulation, which was blocked by the addition of either the ROS scavenger N-acetylcysteine (NAC) or Trolox (Fig. 3D). In contrast, buparlisib alone did not increase ROS accumulation in LNCaP/PIM1 (Fig. 3E). Combined BSO/buparlisib treatment of these cells produced robust ROS accumulation, paralleling the results of animal experiments. These data indicate that increased expression of PIM1 induces resistance to buparlisib in part by stimulating GSH production and suppressing ROS accumulation.
Given the ability of PIM1 to regulate NRF2 levels, potentially controlling the activity of multiple genes which modulate energy production, the effect of PIM1 overexpression on the metabolism of prostate cancer cells was studied. LNCaP/EV or LNCaP/PIM1 cells were labeled with 13C-glucose, and this labeling was chased at 0, 1, and 3 hours. LNCaP/PIM1 had increased carbon flux through both glycolysis and the pentose phosphate shunt. Specifically, there was an increased rate of labeling of glucose 6-phosphate (G6P)/fructose 6-phosphate (F6P) (combined), phosphoenol pyruvate (PEP), 2-phosphogluconate (2PG)/3-phosphogluconcate (3PG) (combined), 6-phosphogluconate (6PG), ribulose 5-phosphate (R5P)/xylulose 5-phosphate (X5P) (combined), and sedoheptulose-7-phosphate (S7P; Supplementary Fig. S3). This increased flux enhanced the mass isotope abundance of NAD(P)H and NADP+ (Fig. 4A). As predicted, 13C abundance in GSH was increased (Fig. 4B). The Seahorse assay was then used to assess OCR and extracellular acidification rate (ECAR). Consistent with the increased flux through the glycolytic pathway, decreased OCR and increased ECAR were evident in LNCaP/PIM1 (Fig. 4C). Moreover, induction of PIM1 resulted in restoration of LDH activity (which was decreased by buparlisib; ref. Fig. 4D). Change in mitochondrial energetics (i.e., OCR) was inhibited by buparlisib treatment but was maintained in LNCaP/PIM1 cells (Fig. 4E). Thus, increased PIM1 appears to stimulate glucose carbon flux, increasing GSH synthesis, potentially through the pentose phosphate pathway, and NAD(P)H production.
Combined inhibition of PIM and AKT blocks the survival and proliferation of prostate cancer cells by inhibiting NRF2 expression through the regulation of mTORC1 activity
Both PC-3 and PC3-LN4 cells with aberrant activation of AKT signaling are relatively insensitive to either buparlisib or AZD5363, but buparlisib cotreatment with PIM447 or AZD1208 (two structurally different PIM inhibitors) blocked their proliferation (Supplementary Figs. S1 and S4A). Genetic inhibition of the AKT pathway by expression of PTEN in PC-3 cells downregulated NRF2 expression (Fig. 5A) and resulted in a mild inhibition of prostate cancer viability. However, this effect was blunted by PIM1 overexpression. Conversely, treatment with PIM447 enhanced PTEN-mediated suppression of prostate cancer viability even in PIM1-overexpressing cells (Fig. 5B). Treatment with the combination of PIM447 and buparlisib increased the accumulation of ROS in the PC3-LN4 cells (Fig. 5C and D; Supplementary Fig. S4B). Consistent with the induction of ROS, NRF2 protein expression was markedly reduced by combination treatment (Fig. 5E). Addition of tert-Butyl hydroperoxide further increased ROS and enhanced the cytotoxicity of buparlisib as well as PIM447 (Supplementary Fig. S4C). In parallel, decreases of cellular GSH and NAD(P)H levels induced by PIM447 were potentiated by buparlisib treatment (Fig. 5F). Forced depletion of all three isoforms of PIM kinases with siRNAs directed at PIM1, 2, and 3 resulted in reduced GSH, NADPH, as well as HMOX1 levels (Supplementary Fig. S4D–S4F), validating that these changes were secondary to PIM inhibition. The increased toxicity of ROS in these cells was reflected in induction of γ-H2AX, a DNA damage marker (Supplementary Fig. S4G). Thus, the combination of PIM and PI3K/AKT inhibitor killed prostate cancer cells through increasing ROS levels.
Although buparlisib treatment of LNCaP cells effectively inhibited mTORC1 activity as judged by phosphorylation of p70S6K, an mTORC1 substrate, PIM1 overexpression abrogated the mTORC1-inhibitory activity of buparlisib (Fig. 5G). When PC-3 or PC3-LN4 cells were treated with buparlisib and PIM447, phosphorylation levels of p70S6K, S6, and 4E-BP1 protein were more strongly inhibited compared with buparlisib alone. Together, these data validate that both the AKT/PI3K pathway and PIM control mTORC1 signaling (Fig. 5H). In LNCaP cells, AZD5363, buparlisib, or Torin1 (an mTOR kinase inhibitor) all blocked mTORC1 signaling and decreased HMOX1 levels, but in PIM1-overexpressing cells, only Torin1 suppressed mTORC1 and HMOX1 expression (Supplementary Fig. S4H). The NQO1-ARE luciferase vector measures the stimulation of transcription by NRF2. When PC3-LN4 cells expressing NQO1-ARE luciferase were treated with Torin1, luciferase activity was inhibited to a similar extent as PIM447 plus buparlisib (Supplementary Fig. S4I). PIM kinase in part regulates mRNA translation by phosphorylating eIF4B on S406 (8). Using CRISPR-Cas9 technology, a knock in of serine to alanine (eIF4B S406A) mutation was produced in human prostate cancer line PC3-LN4. Similar to PIM inhibitor treatment, blocking this eIF4B S406 phosphorylation via a genetic approach markedly inhibited NRF2 and HMOX1 protein expression (Fig. 5I). To examine whether eIF4B is needed for PIM-mediated induction of NRF2 signaling, PIM1-inducible PC-3 cells were transduced with lentivirus of CRISPR-Cas9 eIF4B (sgRNA eIF4B_2) and control vector. Compared with the increased expression of NRF2 and HMOX1 upon Dox-stimulated PIM1 induction in control vector–expressing cells, increases of HMOX1 and NRF2 were blunted in eIF4B-deficient cells even with Dox stimulation (Fig. 5J and K), indicating critical role of eIF4B in PIM1 regulation of NRF2 signaling. Mouse embryonic fibroblasts (MEF) obtained from mice that are genetically deleted in all PIM kinases (triple knockout; TKO) do not phosphorylate eIF4B. These results suggest that inhibition of mTORC1 by PI3K and PIM inhibitors functions to block NRF2 activation, and thus increase ROS production. It is noteworthy that when PIM triple-knockout MEF cells were engineered to express either PIM1, PIM2, or PIM3 (23, 26), expression of each PIM isoform was capable of increasing NRF2 expression (Supplementary Fig. S4J), indicating the overlapping effects of these isoforms on NRF2 signaling.
Because mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system (32), we explored whether increased expression of PIM changes NRF2 levels by partially blocking ubiquitination and subsequent proteasome-mediated protein degradation. To test this possibility, LNCaP cells were cotransfected with PIM1, hemagglutinin (HA)-tagged NRF2, and Flag-tagged ubiquitin plasmids. NRF2 protein was immunoprecipitated using anti-HA agarose beads, and ubiquitylated NRF2 was further detected by immunoblot analysis with an anti-Flag antibody. In LNCaP cells cotransfected with PIM1, a decrease in the level of ubiquitylated NRF2 protein was detected (Fig. 6A), indicating that PIM1 prevents NRF2 ubiquitination. To further examine the role of PIM1, prostate cancer cells were cotransfected with HA-NRF2 and Flag-Ubiquitin followed by the treatment with PIM447 in the presence of MG132, a compound that blocks the degradation of ubiquitylated NRF2. PIM447 treatment induced accumulation of ubiquitinated NRF2 protein (Fig. 6B). Reduction of NRF2 protein levels by PIM447 was blocked by the application of MG132 (Fig. 6C). MG132 also blocked the decrease in NRF2 induced by genetic deletion of PIM (Supplementary Fig. S4K). The half-life of NRF2 protein was decreased by treatment with PIM447 compared with untreated PC3-LN4 control cells from 32.8 to 21.9 minutes (Fig. 6D). This demonstrates the potential ability of PIM kinases to maintain NRF2 protein stability and its function by controlling both the translation and degradation pathways that regulate the levels of NRF2. Therefore, PIM and PI3K-AKT inhibitors synergize to block NRF2 activity, augmenting oxidative stress to suppress tumor growth (Fig. 6E).
Discussion
The PIM protein kinases have been shown to confer resistance to PI3K and AKT inhibitors in part by activating PI3K downstream effectors in an AKT-independent manner (1, 14, 33). PIM inhibition enhances sensitivity to AKT inhibitors, and cancers with mutation in PIK3CA cells often develop increased levels of the PIM kinases (1). Treatment of tumors with agents that inhibit cell surface tyrosine kinases, e.g., MET inhibitors, blocks AKT activation, elevates PIM levels, and ultimately, induces drug resistance (34). Experiments reported here demonstrate the mechanism by which PIM functions to induce resistance to these agents, namely, (1) by increasing NRF2 levels and consequently augmenting the level of ROS scavengers and (2) by stimulating cellular metabolism to drive reduced GSH levels.
The NRF2 transcription factor is an important regulator of cancer survival and proliferation. Redox homeostasis is essential to maintain cellular equilibrium and the survival of cancer cells that have abnormal metabolism (12). Approximately 70% of tumors in biopsies from patients with metastatic prostate cancer harbor aberrant PI3K pathway signaling. This leads to AKT activation, and in this tumor type, HMOX1 plays a pivotal role in antioxidant defense and the regulation of resistance to PI3K inhibition. In prostate cancer cell lines harboring PTEN deletion or mutation, PI3K inhibitor treatment increases ROS levels while decreasing scavenger enzymes. Here, the importance of ROS generation as a means by which these compounds kill prostate cancer was demonstrated by the observation that the expression of HMOX1 blocked the cytotoxicity of the pan-PI3K inhibitor buparlisib, whereas the depletion of ROS scavengers enhanced the activity of this compound. Importantly, even in buparlisib-treated tumor cells, expression of PIM1 elevated NRF2 levels and increased ROS scavengers, e.g., HMOX1, preventing PI3K inhibitor killing of prostate cancer. The combination of a PIM and an AKT inhibitor markedly decreased NRF2 and increased ROS production. Importantly, further increasing ROS generation with tert-Butyl Hydroperoxide stimulated the death of these prostate cancer cells.
Elevated GSH biosynthesis is known to be required for PI3K/AKT-driven resistance to oxidative stress, initiation of tumor spheroids, and anchorage-independent growth (35). The addition of buparlisib to prostate cancer treatment lowered GSH levels along with lowering NAD(P)H, a molecule essential for keeping GSH reduced and inducing oxidative stress. In contrast to buparlisib, PIM1 overexpression enhanced GSH synthesis, abrogating the inhibitory effects of buparlisib. Recent studies have shown that NRF2 regulates genes that are involved in anabolic metabolism. Shown here, in prostate cancer cells, PIM1 facilitated glucose flux into substrates for GSH production and the recycling process. Genes involved in GSH synthesis were not induced by PIM1 when NRF2 was depleted, suggesting that PIM regulation of NRF2 is coordinating the high GSH content in PIM1-overexpressing tumor cells. Synthetic lethal interaction between NRF2 loss and mutant KRAS was detected in pancreatic ductal adenocarcinomas (13). Cells that lack PIM kinase or are treated with a PIM inhibitor were not tolerant of mutant KRAS expression due to lethal ROS accumulation (26), again suggesting that PIM kinases are important regulators of cellular redox signaling. In agreement with other reports (13, 35), AKT inhibitor efficacy was markedly enhanced by cotreatment with the GSH synthesis inhibitor BSO. Here, it is shown that tumor suppression is enhanced by combining BSO and buparlisib. BSO resensitized PIM1-overexpressing tumors to buparlisib both in vitro and in vivo. This suggests that the maintenance of GSH is important to the mechanism by which PIM protects cells from death. Clearly, the regulation of redox homeostasis by PIM1 overlaps with the action of AKT and controls cellular homeostasis.
PIM1 regulates translation by phosphorylating eIF4B, a protein associated with mRNA unwinding, on S406 (8). Shown here, mutation of the PIM phosphorylation site in eIF4B (S406A) markedly decreases NRF2 protein levels, suggesting a mechanism by which PIM controls NRF2 translation. NRF2 exists in complex with KEAP1 via direct protein–protein interactions between the KEAP1 Kelch domain and the ETGE and DLG motifs on the Neh2 domain of NRF2 (36). We further show that PIM kinases appear to partially block the ubiquitination of NRF2, enhancing its half-life, accounting in part for the increased levels of NRF2 when PIM is expressed. However, the mechanism by which PIM regulates NRF2 degradation is not clear. Although there is variability in the level of KEAP1 in prostate cancer cell lines, experiments did not find that PIM1 regulated the levels of NRF2 by modifying either the interaction of NRF2 with KEAP1 or the degradation of KEAP1. NRF2 degradation could be regulated in these cells by phosphorylation on the Neh6 domain of NRF2. Phosphorylation on this site by GSK3 can be recognized by β-TrCP, which acts as a receptor for the SKP1-CUL1-RBX1/ROC1 ubiquitin ligase complex (37) and leads to the degradation of NRF2 in a KEAP1-independent fashion. In human lung cancer A549 cells, inhibition of the PI3K/AKT pathway markedly reduced endogenous NRF2 protein through this mechanism (38).
The mechanism by which PIM regulates NRF2 can have broader impact for molecularly targeted therapy in varied tumor types. Targeting the PI3K signaling pathway has resulted in the development of novel PI3K inhibitors including buparlisib, pictilisib, ZSTK474, dactolisib, apitolisib, and omipalisib. A PI3K-α–selective inhibitor alpelisib is currently in clinical development for breast cancer therapy in a phase III clinical trial (NCT02437318) and phase II study for treatment of head and neck squamous cell carcinoma (NCT02051751). In 2014, the p110δ-specific inhibitor idelalisib was approved in the United States and Europe as the first-in-class PI3K inhibitor for use in the treatment of chronic lymphocytic leukemia and follicular lymphoma. As well, copanlisib with activity against the PI3K-α and -δ isoforms was recently approved by the FDA for treatment of patients with relapsed follicular lymphoma. However, in prostate cancer although mutations activate the PI3K pathway, inhibitors targeting this pathway have not shown activity. Our findings in prostate cancer models that combining PI3K inhibitors with a PIM inhibitor enhances the induction of cell death by markedly increasing ROS generation may provide molecular basis to overcome the limited efficacy of PI3K-targeted therapies. The therapeutic vulnerabilities caused by redox regulation in cancer suggest the use of PIM inhibitors as an additional treatment modality.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: J.H. Song, N.A. Warfel, A.S. Kraft
Development of methodology: J.H. Song, L.A. Luevano, K. Okumura, V. Olive, S.M. Black
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.H. Song, N. Singh, L.A. Luevano, S.M. Black, D.W. Goodrich
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.H. Song, N. Singh, L.A. Luevano, A.S. Kraft
Writing, review, and/or revision of the manuscript: J.H. Song, N. Singh, S.K.R. Padi, K. Okumura, V. Olive, N.A. Warfel, D.W. Goodrich
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.H. Song, N. Singh, L.A. Luevano, S.K.R. Padi, A.S. Kraft
Study supervision: J.H. Song, A.S. Kraft
Other (development of key experimental models): D.W. Goodrich
Acknowledgments
This research was supported by University of Arizona Cancer Center support grant NIH P30CA023074, NIH award R01CA173200, DOD award W81XWH-12-1-0560 (to A.S. Kraft), and American Lung Association Award LCD-504131 (to N.A. Warfel). The authors thank Novartis Oncology for providing PIM447. The UACC Shared Resources provided support for histologic and tissue staining, microarray, and flow cytometry analysis.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.