Aberrant expression of the kinase IKKϵ in pancreatic ductal adenocarcinoma (PDAC) has been associated with poor prognosis. In this study, we define a pathobiologic function for IKKϵ in reprogramming glucose metabolism and driving progression in PDAC. Silencing IKKϵ in PDAC cells, which overexpressed it endogenously, was sufficient to reduce malignant cell growth, clonogenic potential, glucose consumption, lactate secretion, and expression of genes involved in glucose metabolism, without impacting the basal oxygen consumption rate. IKKϵ silencing also attenuated c-Myc in a manner associated with diminished signaling through an AKT/GSK3β/c-MYC phosphorylation cascade that promoted MYC nuclear accumulation. In an orthotopic mouse model, IKKϵ-silenced PDAC exhibited a relative reduction in glucose uptake, tumorigenicity, and metastasis. Overall, our findings offer a preclinical mechanistic rationale to target IKKϵ to improve the therapeutic management of PDAC in patients. Cancer Res; 76(24); 7254–64. ©2016 AACR.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a 5-year survival rate of about 8% after initial diagnosis (1). It is expected to overtake breast malignancy this year as the third leading cause of cancer-related deaths in the United States with an estimated 53,070 new diagnoses and 41,780 deaths, and may actually become the second by 2020 if similar trends continue (1, 2). Over the years, significant progress has been made in our understanding of the genetics of PDAC (3); however, these seminal advancements have not helped much in the development of an effective treatment strategy for this lethal malignancy. As a consequence, search for novel, functionally relevant molecular targets continues, so that effective, mechanism-based approaches for its therapy and management can be formulated.

Inhibitor of kappa kinase subunit-epsilon (IKKϵ) is an important member of the IKK family along with four other, distinct yet closely related, members (IKKα, IKKβ, IKKγ, and NAK). IKKϵ plays a central role in innate immunity by inducing NF-κB- and IFN regulatory factor (IRF)-dependent gene transcription of proinflammatory cytokines and interferons (4). It is expressed at basal levels in a subset of tissues involved in immune function, and can be readily induced in a variety of cell- and tissue types upon external stimuli (5). Interestingly, IKKϵ has also been shown to regulate energy balance in high-fat diet–induced obesity (6) and recognized to possess oncogenic properties in breast cancer (7) with some later reports in other cancers as well (8, 9). In many cancer cases, including PDAC, an upregulation of IKKϵ, even in the absence of gene amplification, has been reported and associated with poor clinical outcome (7, 9, 10). However, we lack direct evidence for its oncogenic activity in PDAC along with complete lack of an in-depth understanding of involved molecular pathways.

Cancer cells remain under constant demand for energy and building blocks to ensure their continued, rapidly proliferative development. As a result, they adapt to glycolytic metabolism, even when oxygen is not a limiting factor, to meet their demands for quick energy (ATPs) and metabolic intermediates that serve as building blocks for rapidly dividing cancer cells (11). This shift provides added advantage to the tumor cells, that is, the ability to thrive independently of oxygen diffusion that would otherwise be a limiting factor for rapidly growing tumors (12). Indeed, mounting evidence continues to associate enhanced aerobic glycolysis to the etiology and malignant progression of several cancers, including pancreatic malignancy (13). This metabolic shift, in general, is mediated through aberrant activation of oncogenic transcription factors, leading to altered expression of genes involved in glucose import and metabolism (14). c-MYC serves as a “master regulator” of growth and cellular metabolism pathways, and its aberrant activation is facilitated at multiple levels (15–17). Emerging clinical and experimental data also support its role in PDAC pathobiology (10).

This study provides first evidence for a link between IKKϵ and c-MYC oncoprotein. We demonstrate that IKKϵ regulates nuclear retention and stabilization of c-MYC through a cascade of signaling events. We further identify a novel role of IKKϵ in regulating glucose metabolism in PDAC, at least in part, through its c-MYC–mediated regulation of metabolic gene expression. IKKϵ overexpression is also shown to promote growth and metastasis of PDAC cells, thus establishing it as an important molecular target for clinical management.

Cell lines and tissue samples

The human pancreatic cell lines were obtained and maintained as previously described (18). All the cell lines were tested intermittently and determined to be free from mycoplasma, and authenticated by either in-house or commercial (Genetica DNA Laboratories) short-tandem repeats genotyping. Normal and tumor pancreatic tissue specimens were obtained through the Southern Division of Cooperative Human Tissue Network under an Institutional Review Board–approved protocol.

Antibodies

Antibodies used were: anti-IKKε, -c-MYC (rabbit monoclonal), -phospho-c-MYCS62, -phospho-c-MYCT58 (rabbit polyclonal), -ubiquitin (mouse monoclonal; Abcam); -phospho-AktT308 (rabbit monoclonal), -phospho-AktSer473 (rabbit polyclonal; Cell Signaling Technologies); -GSK3β, -phospho-GSK3βSer9, -Akt (rabbit monoclonal; Epitomics); -LaminA, (mouse monoclonal), -α-tubulin (rabbit polyclonal; Santa Cruz Biotechnology). Anti-β-actin–HRP–conjugated (mouse monoclonal) antibody was from Sigma-Aldrich. All secondary antibodies were from Santa Cruz Biotechnology.

Transfections and treatments

Generation of stable IKKϵ-knockdown and control cell lines was done in IKKϵ-overexpressing MiaPaCa and Colo357 cells by transfection of IKBKE-shRNA-pGFP-B-RS or the control-plasmid, Scr-shRNA-pGFP-B-RS (Origene), respectively, using X-tremeGENE HP DNA Transfection Reagent (Roche) as per the manufacturer's instructions. Transfectants were selected using blasticidin (2 μg/mL MiaPaCa and 20 μg/mL Colo357) and assessed for IKKϵ expression using immunoblotting. For transient knockdown, cells were cultured in 6-well plates and transfected with 100 nmol/L of nontarget or ON-TARGETplus SMARTpool IKBKE-targeting siRNAs (Dharmacon) using DharmaFECT (Dharmacon) according to the manufacturer's instruction. Cycloheximide (50 μmol/L; Sigma-Aldrich) and MG132 (10 μmol/L; Sigma-Aldrich) were used to inhibit protein synthesis or proteasome-degradation machinery, respectively. Cells were treated with GSK3β inhibitor LiCl (40 mmol/L; Sigma-Aldrich) for 6 hours or transiently transfected with constitutively active Akt (pcDNA3-HA PKB T308D S473D, plasmid number 14751; Addgene) mutant or control plasmids to dissect the roles of GSK3β and Akt, respectively.

Immunoblot analysis

Total protein from PDAC cells was isolated in NP-40 lysis buffer supplemented with phosphatase and protease inhibitors (Roche), and estimated using DC Protein-Assay Kit (Bio-Rad). Protein samples (60–80 μg, unless noted) were resolved on SDS-PAGE and subjected to immunoblot analysis as described previously (18, 19). Band intensities were quantitated using ImageJ software.

Growth kinetics assay

Growth rate and population-doubling time (PDT) were determined by counting number of viable cells using the Trypan blue dye exclusion on the Countess Automated Cell Counter (Life Technologies) every day for 8 days, as described previously (18).

Clonogenicity assays

Anchorage-dependent and anchorage-independent clonogenicity assays were carried out as described previously (20).

qPCR and Ingenuity Pathway Analysis

RNA isolation, cDNA synthesis, and qPCR were performed as described previously (20) using primers listed in Supplementary Table S2. The altered genes (fold-change ≥ 1.5; P ≤ 0.05) were subjected to Ingenuity Pathway Analysis (IPA) to identify putative upstream regulator.

Luciferase assay

Control or IKKϵ-silenced PDAC cells were transfected with either a negative control or c-MYC–responsive luciferase-promoter-reporter plasmid (Cignal MYC Reporter Assay Kit, SABiosciences), and assayed as per the manufacturer's protocol.

Nuclear and cytoplasmic fractionation

Cytoplasmic and nuclear extracts were prepared using Nuclear-Extract Kit (Active Motif) following the manufacturer's instructions.

Coimmunoprecipitation analysis

Coimmunoprecipitation was performed using c-MYC–specific antibody as described previously (21).

Glucose uptake and lactate production assays

Glucose and lactate concentration in the culture media was determined using the Glucose and Lactate Assay Kit (Biovision) as per manufacturer's instructions. To measure glucose uptake, cells were first incubated in glucose-free, FBS-free media for 6 hours followed by incubation with a glucose-free DMEM supplemented with 100 μmol/L of fluorescent d-glucose derivative, 2-NBDG (Invitrogen) for 3 hours, and analyzed by fluorescence imaging or flow cytometry using FACS AriaII (BD Biosciences).

Measurement of extracellular acidification rate and oxygen consumption rate

Basal rate of glycolysis and oxidative phosphorylation was determined by measuring the extracellular acidification (ECAR) and the oxygen consumption rates (OCR) using the Seahorse-XF24 Analyzer (Seahorse Bioscience) as per the manufacturer's instructions. Briefly, 4 × 104 cells per well were seeded in XF24 cell culture microplates in a two-step process and incubated at 37°C for 24 hours. Subsequently, culture media was changed to XF Assay Medium (supplemented with 5 mmol/L glucose), and plates loaded into the XF24 analyzer to record the data.

Assessment of tumorigenicity and glucose uptake in vivo

Animal studies were conducted under protocol approved by Institutional Animal Care and Use Committee. Luciferase-tagged IKKϵ-knockdown or control cells (1 × 106 cells/50 μL) were injected into the pancreas of immunocompromised mice (Harlan Laboratories; 10 mice/group). After 1 week, tumor growth was monitored every third day by palpation and weekly by noninvasive in vivo imaging. For glucose uptake, mice were injected intraperitoneally with 100 μL of XenoLight RediJect 2-DeoxyGlucosone (2-DG; Perkin Elmer) 24 hours prior to final imaging (28 days postimplantation) and epi-fluorescence recorded using Xenogen-IVIS-cooled CCD optical system (IVIS Spectrum) next day. To image tumors, mice were given intraperitoneal injections of d-Luciferin (150 mg/kg body weight) and bioluminescence recorded using the Xenogen-IVIS-cooled CCD optical system. After euthanasia, primary tumors were resected and mice imaged for the detection of metastases. Final measurements of tumor weight and volumes were also made from resected xenografts.

WST-1 proliferation assay

Cells (3 × 103/well) were seeded in 96-well plates and growth examined using WST-1 Assay Kit (Roche) and analyzed as described previously (19).

Statistical analysis

The experiments were conducted in triplicates and repeated at least three times. Wherever appropriate, the data were also subjected to unpaired two-tailed Student t test. P ≤ 0.05 was considered significant.

IKKϵ is overexpressed and associated with increased growth and clonogenicity of PDAC cells

We first analyzed IKKϵ expression in a set of malignant (n = 21) and non-neoplastic (n = 7) pancreas. Similar to a published report (10), we observed an overexpression of IKKϵ in 81% cases of malignant pancreas, whereas no (n = 5) or very low (n = 2) expression was detected in non-neoplastic cases (Supplementary Fig. S1A). In search of model cell lines for functional studies, we analyzed IKKϵ expression in a panel of 14 PDAC cell lines, of which 12 expressed high-to-moderate levels, whereas 2 had low/null expression (Supplementary Fig. S1B). Considering that IKKϵ-overexpressing cells would likely have IKKϵ-dependent growth mechanisms, we selected MiaPaCa and Colo357 cells that carry most common PDAC genetic aberrations (KRASmut, TP53mut, CDKN2Adel/inactive) and have been well characterized for their aggressiveness and metastatic potential (22, 23) for functional studies. IKKϵ expression was silenced by stable transfection of three different IKBKE-targeted shRNA expression constructs (ShIKKϵ#1–3). Control cells were generated by stable transfections with nontargeted scrambled sequence (Scr) expression construct. Stable silencing of IKKϵ was analyzed by immunoblotting in polyclonal populations from individual constructs (Fig. 1A). As ShIKKϵ plasmid #1 and #3 transfectants exhibited most potent reduction in IKKϵ, compared with control, they were combined to generate pooled population (ShIKKϵ; Fig. 1B). IKKϵ inhibition resulted in significant growth reduction in MiaPaCa (∼45.4%) and Colo357 (∼42.4%) cells on the eighth day of growth kinetics (Fig. 1C) due to increase in their PDT calculated during exponential growth phase (96–144 hours). Silencing of IKKϵ prolonged PDT from approximately 36.46 and 21.14 hours to approximately 45.71 and ∼30.37 hours in MiaPaCa–ShIKKϵ and Colo357–ShIKKϵ cells, respectively, relative to their controls (Supplementary Table S2). Moreover, when seeded at low density, we observed a significant decrease (P < 0.05) in the plating efficiency of MiaPaCa–ShIKKϵ (∼2.8-fold) and Colo357–ShIKKϵ (∼3.2-fold) cells as compared with their controls (Fig. 1D). A significant (P < 0.05) decrease (∼3.5- and ∼3.9-fold, respectively) was also recorded in anchorage-independent clonogenic potential (an in vitro measure of tumorigenecity) of MiaPaCa–ShIKKϵ and Colo357–ShIKKϵ cells relative to their controls (Fig. 1E). To further confirm the role of IKKϵ in pancreatic cancer cell growth, we also ectopically overexpressed IKKϵ in BxPC3 cells that has its low endogenous expression (Supplementary Fig. S2A). IKKϵ overexpression led to increase in growth of BxPC3 cells by approximately 37.94% on the eighth day of growth kinetics due to decrease in doubling time (Supplementary Fig. S2B).

Figure 1.

IKKϵ supports the growth and clonogenicity of PDAC cells. A, Total protein isolated from stably transfected PDAC cells was examined for IKKϵ expression by immunoblotting. B, Clonal population of transfectants emerging from IKKϵ-targeting shRNA constructs #1 and #3, which consistently produced maximum silencing of IKKϵ, were pooled (ShIKKϵ), propagated, and monitored for IKKϵ expression by immunoblot assay. β-Actin was used as loading control. C, Growth kinetics of IKKϵ-silenced clonal and pooled population was studied relative to their controls for 8 days using the Trypan blue exclusion assay. D, Plating efficiency was measured by seeding the PDAC cells at low density. E, Anchorage-independent colony formation was measured as an in vitro measure of tumorigenic ability of control and IKKϵ-silenced PDAC cells. Data are presented as mean ± SD, n = 3; **, P ≤ 0.05.

Figure 1.

IKKϵ supports the growth and clonogenicity of PDAC cells. A, Total protein isolated from stably transfected PDAC cells was examined for IKKϵ expression by immunoblotting. B, Clonal population of transfectants emerging from IKKϵ-targeting shRNA constructs #1 and #3, which consistently produced maximum silencing of IKKϵ, were pooled (ShIKKϵ), propagated, and monitored for IKKϵ expression by immunoblot assay. β-Actin was used as loading control. C, Growth kinetics of IKKϵ-silenced clonal and pooled population was studied relative to their controls for 8 days using the Trypan blue exclusion assay. D, Plating efficiency was measured by seeding the PDAC cells at low density. E, Anchorage-independent colony formation was measured as an in vitro measure of tumorigenic ability of control and IKKϵ-silenced PDAC cells. Data are presented as mean ± SD, n = 3; **, P ≤ 0.05.

Close modal

Silencing of IKKϵ inhibits glycolytic metabolism in prostate cancer

As a defined hallmark, tumor cells sustain their rapid growth by shifting to glycolytic metabolism to ensure quick and sufficient supply of energy and macromolecular precursors (11, 14). Therefore, we evaluated the influence of IKKϵ silencing on glucose metabolism in PDAC cells. First, consumption of glucose and the lactate release was measured from the used culture media of control and IKKϵ-silenced PDAC cells, which demonstrated substantial attenuation of glucose consumption (Fig. 2A) and lactate secretion (Fig. 2B) upon IKKϵ silencing. The influence on glucose uptake in a cell-autonomous manner in control and IKKϵ-silenced cells was further investigated using the fluorescent glucose analogue 2-NBDG, which was imaged by fluorescence microscopy or quantified using flow cytometry upon incorporation into the cells. These experiments were carried out after transient silencing of IKKϵ in MiaPaCa and Colo357 cell lines using ON-TARGETplus SMARTpool IKBKE-targeting siRNAs or nontargeting control siRNAs, as stable lines expressed GFP, which could hinder 2-NBDG signal detection. IKKϵ silencing by siRNA was determined at 72 hours by immunoblotting (Supplementary Fig. S3). In line with the above observation, cellular accumulation of 2-NBDG was significantly less in IKKϵ-silenced PDAC cells compared with their control cells (Fig. 2C) as quantitative analysis by flow cytometry revealed a reduction in the mean fluorescence intensity by 23.62% and 33.32% in MiaPaCa–SiIKKϵ and Colo357–SiIKKϵ cells, respectively, relative to their controls (Fig. 2D). To get further insight into altered metabolic phenotype, we monitored basal glycolytic metabolism and oxidative phosphorylation by extracellular acidification rate (ECAR) and OCR using Seahorse-XF Extracellular-Flux Analyzer. ECAR is a surrogate measure of glycolysis and an alternate measure for lactate secretion, whereas OCR measures the changes in dissolved oxygen concentration. We observed a significant decrease in the glycolytic activity of IKKϵ-silenced cells relative to control cells; however, no substantial change in oxygen consumption was recorded (Fig.,2E and F). In concordance, overexpression of IKKϵ in BxPC3 cells enhanced glucose uptake and lactate efflux, which was also reflected in elevated ECAR (Supplementary Fig. S4). This suggests that IKKϵ silencing impacts glycolytic phenotype only without having noticeable effect on the electron transport system machinery.

Figure 2.

IKKϵ silencing suppresses glucose uptake and consumption in PDAC cells. A and B, Glucose uptake (A) and lactate efflux (B) was measured in the used culture media and normalized to cell counts. The data are depicted as percent change in IKKϵ-silenced cells relative to their respective controls. C and D, PDAC cells were transfected with nontargeting control (NT) or IKBKE-targeting (SiIKKϵ) siRNAs. After 72 hours, cells were cultured in glucose-free FBS-free media for 6 hours and further incubated with glucose-free media supplemented with 100 μmol/L 2-NBDG for 3 hours. Thereafter, either the cells were visualized under fluorescent microscope and photographed (C) or subjected to flow cytometry analysis (D). Representative images are from independent experiments. E and F, To examine basal ECAR and OCR, 4 × 104 cells per well were seeded in XF24 cell culture microplates and incubated at 37°C overnight. Next day, culture media was replaced with XF Assay Medium (supplemented with 5 mmol/L glucose) and plates loaded into the XF24 analyzer. Data are presented as mean ± SD, n = 3; **, P ≤ 0.05.

Figure 2.

IKKϵ silencing suppresses glucose uptake and consumption in PDAC cells. A and B, Glucose uptake (A) and lactate efflux (B) was measured in the used culture media and normalized to cell counts. The data are depicted as percent change in IKKϵ-silenced cells relative to their respective controls. C and D, PDAC cells were transfected with nontargeting control (NT) or IKBKE-targeting (SiIKKϵ) siRNAs. After 72 hours, cells were cultured in glucose-free FBS-free media for 6 hours and further incubated with glucose-free media supplemented with 100 μmol/L 2-NBDG for 3 hours. Thereafter, either the cells were visualized under fluorescent microscope and photographed (C) or subjected to flow cytometry analysis (D). Representative images are from independent experiments. E and F, To examine basal ECAR and OCR, 4 × 104 cells per well were seeded in XF24 cell culture microplates and incubated at 37°C overnight. Next day, culture media was replaced with XF Assay Medium (supplemented with 5 mmol/L glucose) and plates loaded into the XF24 analyzer. Data are presented as mean ± SD, n = 3; **, P ≤ 0.05.

Close modal

IKKϵ alters the expression of genes of glucose metabolic pathway that converge at c-MYC

To identify the altered gene expression associated with shift in glucose metabolism, we employed a qPCR-based custom array that included 80 genes involved in glucose metabolism. A total of 33 genes were found to be dysregulated upon IKKϵ silencing including those involved in glucose transport, glycolysis, gluconeogenesis, TCA cycle, pentose phosphate pathway, and lactate transport (Fig. 3A). Gene expression dataset was subjected to the IPA software in search for a putative upstream transcriptional regulator mediating the effect of IKKϵ silencing. In silico analysis suggested c-MYC to be a candidate upstream regulator of altered gene expression (Fig. 3B). To confirm the predicted inhibition of c-MYC, we examined its transcriptional activity using a c-MYC–responsive promoter reporter system and observed significant abrogation (79% and 63%, P < 0.05) in luciferase activity of IKKϵ-silenced MiaPaCa and Colo357 cells, respectively, relative to their controls (Fig. 3C). This correlated with a reduction in total c-MYC protein (Fig. 3D) without any significant change in its transcripts levels (Fig. 3E). Interestingly, subcellular fractionation demonstrated greater nuclear accumulation of c-MYC in IKKϵ-expressing control cells, while relatively greater cytoplasmic levels of c-MYC were detected in IKKϵ-silenced cells (Fig. 3F). In line with these observations, ectopic expression of IKKϵ in BxPC3 cells also elevated c-MYC protein level along with a greater nuclear accumulation (Supplementary Fig. S5). These cells, however, sustained significant c-MYC in the cytoplasm, thus suggesting the involvement of additional cell type and/or context-dependent mechanism(s) in regulation of c-MYC by IKKϵ.

Figure 3.

IKKϵ alters the expression of genes encoding glucose-metabolizing enzymes and regulates c-MYC expression and subcellular localization. A, Expression of genes involved in glucose metabolism was measured by qRT-PCR. Data shown as fold change in IKKϵ-silenced cells relative to control. B, Differential gene expression dataset was subjected to IPA that predicted c-MYC as a potential upstream regulator. C, Transcriptional activity of c-MYC was measured using luciferase promoter-reporter assay. Data (mean ± SD, n = 3) are presented as fold change in normalized luciferase activity; **, P ≤ 0.05. Expression of c-MYC in IKKϵ knockdown and control cells was examined at protein (D) and transcript levels (E) by immunoblot and qRT-PCR assays, respectively. ACTB (for mRNA) and β-actin (for protein) were used as internal controls. F, Expression of c-MYC in cytoplasmic and nuclear fractions was examined by immunoblot analysis. Lamin A and α-tubulin were used as loading controls for nuclear and cytoplasmic fractions, respectively.

Figure 3.

IKKϵ alters the expression of genes encoding glucose-metabolizing enzymes and regulates c-MYC expression and subcellular localization. A, Expression of genes involved in glucose metabolism was measured by qRT-PCR. Data shown as fold change in IKKϵ-silenced cells relative to control. B, Differential gene expression dataset was subjected to IPA that predicted c-MYC as a potential upstream regulator. C, Transcriptional activity of c-MYC was measured using luciferase promoter-reporter assay. Data (mean ± SD, n = 3) are presented as fold change in normalized luciferase activity; **, P ≤ 0.05. Expression of c-MYC in IKKϵ knockdown and control cells was examined at protein (D) and transcript levels (E) by immunoblot and qRT-PCR assays, respectively. ACTB (for mRNA) and β-actin (for protein) were used as internal controls. F, Expression of c-MYC in cytoplasmic and nuclear fractions was examined by immunoblot analysis. Lamin A and α-tubulin were used as loading controls for nuclear and cytoplasmic fractions, respectively.

Close modal

c-MYC downregulation upon IKKϵ silencing is caused by its nuclear export and subsequent proteasomal degradation

Differential subcellular distribution of c-MYC besides its overall repression at the protein level prompted us to investigate the underlying molecular mechanism(s). Phosphorylation at conserved serine-62 (S62) residue of c-MYC governs the activation and nuclear import of c-MYC and subsequent phosphorylation of threonine-58 (T58) residue leads to its nuclear export followed by degradation (15–17). Therefore, we examined their levels in IKKϵ-overexpressing and -silenced PDAC cells, which demonstrated a decrease in phospho-c-MYCS62 in IKKϵ-silenced cells (Fig. 4A) likely due to diminished total c-MYC protein content. However, despite reduced levels of total c-MYC, IKKϵ-silenced PDAC cells had significantly increased levels of phospho-c-MYCT58, whereas negligible c-MYCT58 phosphorylation was detected in control cells of both PDAC lines (Fig. 4A). A similar correlation between differential c-MYC phosphorylation and IKKϵ was also observed upon forced IKKϵ expression in BxPC3 cells (Supplementary Fig. S5). Because c-MYCT58 phosphorylation is known to promote its nuclear export followed by ubiquitin (Ub)-mediated proteasomal degradation (15–17), we next examined the effect of MG-132 (proteasome inhibitor) treatment on c-MYC levels. To correct the differences in initial protein levels, we adjusted protein loading as mentioned in the figure legend. IKKϵ-silenced PDAC cells exhibited a time-dependent increase in total c-MYC levels upon MG-132 treatment (Fig. 4B, top). Similar observation was also recorded in MG-132–treated control cells (Fig. 4B, bottom); however, the rate of c-MYC accumulation in these cells was relatively slower compared with IKKϵ-silenced cells. To confirm that c-MYC stabilization was a result of decreased degradation of ubiquitinated-c-MYC, we performed immunoprecipitation using anti-c-MYC antibody followed by immunoprobing with anti-Ub antibody. An accumulation of Ub-c-MYC upon MG132 treatment was observed in both control and IKKϵ-silenced cells; however, the latter exhibited elevated levels (Fig. 4C). To measure the overall impact of IKKϵ silencing on the stability of c-MYC, we measured its turn-over after blocking protein synthesis by cycloheximide (CHX; Fig. 4D). Differences in initial protein levels were corrected by adjusting protein loading as mentioned in the figure legend. Subsequent analyses based on the densitometry measurements of c-MYC signals predicted half-life of c-MYC to be about 41 and 30 minutes in MiaPaCa and Colo357 cells, respectively, whereas it was decreased to approximately 21 and 16 minutes in IKKϵ-silenced MiaPaCa and Colo357 cells, respectively (Supplementary Fig. S6).

Figure 4.

IKKϵ-induced c-MYC expression and localization is controlled through its inhibition of phosphorylation-mediated nuclear export and subsequent degradation. A, Phosphorylated c-MYCS62 and c-MYCT58 were analyzed by immunoblot using specific antibodies. B, Cells were treated with the proteasome inhibitor (MG132, 10 μmol/L) for the indicated time period, total protein isolated, and effect on c-MYC expression determined by immunoblot analysis. We used 60 and 25 μg protein from IKKϵ-silenced and control cells, respectively, to correct for differences in initial protein levels. β-Actin was used as loading control. C, Equal amount (500 μg) of protein from Scr and ShIKKϵ PDAC cells untreated or treated with MG132 (10 μmol/L, 30 minutes) was subjected to immunoprecipitation with anti-c-MYC antibody, followed by immunoblot with anti-Ub antibody. D, To monitor the turnover of c-MYC protein, cells were treated with cycloheximide (CHX; 50 μmol/L), neo-protein synthesis inhibitor, for the indicated time intervals. Thereafter, total protein was isolated and changes in c-MYC expression monitored by immunoblotting. Considering differences in c-MYC levels between control and IKKϵ-silenced cells, we used different amounts (60 and 150 μg, respectively) to keep initial signal at near-similar intensity. , the rate of total c-MYC accumulation; , the rate of c-MYC degradation based on the densitometry of the data presented.

Figure 4.

IKKϵ-induced c-MYC expression and localization is controlled through its inhibition of phosphorylation-mediated nuclear export and subsequent degradation. A, Phosphorylated c-MYCS62 and c-MYCT58 were analyzed by immunoblot using specific antibodies. B, Cells were treated with the proteasome inhibitor (MG132, 10 μmol/L) for the indicated time period, total protein isolated, and effect on c-MYC expression determined by immunoblot analysis. We used 60 and 25 μg protein from IKKϵ-silenced and control cells, respectively, to correct for differences in initial protein levels. β-Actin was used as loading control. C, Equal amount (500 μg) of protein from Scr and ShIKKϵ PDAC cells untreated or treated with MG132 (10 μmol/L, 30 minutes) was subjected to immunoprecipitation with anti-c-MYC antibody, followed by immunoblot with anti-Ub antibody. D, To monitor the turnover of c-MYC protein, cells were treated with cycloheximide (CHX; 50 μmol/L), neo-protein synthesis inhibitor, for the indicated time intervals. Thereafter, total protein was isolated and changes in c-MYC expression monitored by immunoblotting. Considering differences in c-MYC levels between control and IKKϵ-silenced cells, we used different amounts (60 and 150 μg, respectively) to keep initial signal at near-similar intensity. , the rate of total c-MYC accumulation; , the rate of c-MYC degradation based on the densitometry of the data presented.

Close modal

c-MYC destabilization upon IKKϵ silencing is mediated through Akt repression–induced GSK3β activation

Considering a role of GSK3β in c-MYCT58 phosphorylation (15), we examined its activation status in IKKϵ-expressing and -silenced PDAC cells. GSK3βS9 phosphorylation, which is known to cause its inactivation (24), was decreased in both IKKϵ-silenced PDAC cell lines, without any appreciable changes in total GSK3β (Fig. 5A). When IKKϵ-silenced PDAC cells were treated with LiCl, an inhibitor of GSK3β (25), it restored total c-MYC levels to significant extent, which correlated with decreased phospo-c-MYCT58 and enhanced phospo-GSK3βS9 levels (Fig. 5B). Furthermore, LiCl treatment also enhanced the nuclear retention of c-MYC in IKKϵ-silenced PDAC cells (Fig. 5B). We next investigated whether IKKϵ was directly able to phosphorylate GSK3β through its Ser/Thr kinase activity as reported for GSK3α (26). However, no interaction between IKKϵ and GSK3β was observed in reciprocal coimmunoprecipitation assays (data not shown). Hence, we focused our attention to Akt, which is known to inactivate GSK3β through its S9 phosphorylation (27). Moreover, IKKϵ can also directly phosphorylate Akt at both T308 and S473, independent of PI3K and mTORC2 activities, respectively (28, 29). A significant reduction in AktT308/S473 levels was observed in immunoblot analyses in IKKϵ-silenced PDAC cells (Fig. 5C). Similarly, ectopic expression of IKKϵ in BxPC3 cells promoted Akt activation as evidenced by phosphorylation at Thr-308 and Ser-473 sites, and thus resulting in Ser-9 phosphorylation of GSK3β (Supplementary Fig. S7). Thus, to further explore the role of activated Akt, we expressed its constitutively active mutant form (T308D-S473D; CA-Akt) in IKKϵ-silenced cells through transient transfection. Upon ectopic activation of Akt, significant upregulation of c-MYC protein was detected that correlated with phospho-GSK3βS9 and reduced c-MYCT58 levels (Fig. 5D). Thereafter, we ascertained a role of IKKϵ/Akt/GSK3β signaling axis in glucose metabolism and expression of involved gene targets. First, the effect of LiCl and CA-Akt on relative mRNA expression of some randomly selected genes (HK2, LDHA, SLC2A3, and SLC16A1) was examined followed by glucose uptake measurements. LiCl and CA-Akt activity rescued the gene expression either partially (SLC2A3 and SLC16A1) or almost completely (HK2 and LDHA) in IKKϵ-silenced cells relative to that in control cells (Fig. 5E). Similarly, a significant increase in glucose uptake and lactate efflux was also observed upon LiCl treatment and CA-Akt expression in IKKϵ-silenced cells, which correlated with their enhanced growth (Fig. 5F).

Figure 5.

Subcellular localization and stabilization of c-MYC is governed through IKKϵ/Akt/GSK3β axis. A, GSK3βS9 phosphoryation was determined by immunoblotting using specific antibodies. B, PDAC cells were treated with GSK3β inhibitor, LiCl (40 mmol/L, 6 hours), followed by total protein isolation and immunoblotting (top). Subsequently, cytoplasmic and nuclear levels of c-MYC were also measured following LiCl treatment (bottom). C, Determination of phospho-AktT308/S473 by immunoblotting suggested their reduced expression in IKKϵ-silenced PDAC cells. D, Cells were transfected with constitutively active-Akt (CA-Akt) or control plasmid, followed by measurement of c-MYC, c-MYCT58, pGSK3βS9, and total GSK3β by immunoblotting. E, In parallel experiments, effect of LiCl treatment and CA-Akt transfection was investigated on mRNA levels of HK2, LDHA, SLC2A3, and SLC16A1 by qRT-PCR. F, Glucose uptake (top) and lactate efflux (middle), and growth recovery on day 3 and 5 by WST1 assay (bottom). Bars, mean ± SD (n = 3); **, P ≤ 0.05. β-Actin was used as a loading control for total protein, and Lamin A and α-tubulin were used as controls for nuclear and cytoplasmic fractions, respectively. qPCR data were normalized with ACTB expression.

Figure 5.

Subcellular localization and stabilization of c-MYC is governed through IKKϵ/Akt/GSK3β axis. A, GSK3βS9 phosphoryation was determined by immunoblotting using specific antibodies. B, PDAC cells were treated with GSK3β inhibitor, LiCl (40 mmol/L, 6 hours), followed by total protein isolation and immunoblotting (top). Subsequently, cytoplasmic and nuclear levels of c-MYC were also measured following LiCl treatment (bottom). C, Determination of phospho-AktT308/S473 by immunoblotting suggested their reduced expression in IKKϵ-silenced PDAC cells. D, Cells were transfected with constitutively active-Akt (CA-Akt) or control plasmid, followed by measurement of c-MYC, c-MYCT58, pGSK3βS9, and total GSK3β by immunoblotting. E, In parallel experiments, effect of LiCl treatment and CA-Akt transfection was investigated on mRNA levels of HK2, LDHA, SLC2A3, and SLC16A1 by qRT-PCR. F, Glucose uptake (top) and lactate efflux (middle), and growth recovery on day 3 and 5 by WST1 assay (bottom). Bars, mean ± SD (n = 3); **, P ≤ 0.05. β-Actin was used as a loading control for total protein, and Lamin A and α-tubulin were used as controls for nuclear and cytoplasmic fractions, respectively. qPCR data were normalized with ACTB expression.

Close modal

IKKϵ promotes glucose uptake and tumorigenicity of PDAC cells in an orthotopic mouse model

To assess the functional relevance of IKKϵ in vivo, we injected luciferase-tagged MiaPaCa–Scr/MiaPaCa–ShIKKϵ cells directly into the pancreas of mice, and monitored tumor growth every alternate day by palpation and weekly by noninvasive bioluminescence imaging. A significantly greater fluorescent signal was detected in all mice from MiaPaCa–Scr group as compared with that from MiaPaCa–ShIKKϵ group, indicating higher glucose uptake by the tumors (Fig. 6A). To normalize the glucose uptake with tumor size, we also performed bioluminescent imaging of tumors following d-luciferin injection (Fig. 6B). Normalized fluorescence signal also suggested significantly greater glucose uptake in IKKϵ-overexpressing MiaPaCa–Scr cells compared with IKKϵ-silenced MiaPaCa–ShIKKϵ cells (Fig. 6C). End-point measurement also confirmed significant decrease in tumor growth in IKKϵ-silenced group. Average weight and volume of the tumors developed in control group were recorded to be 1.71 ± 0.05 g and 866 ± 64.86 mm3 as compared with 0.303 ± 0.01 g and 259.9 ± 19.42 mm3, respectively, in IKKϵ-silenced group (Fig. 6D). Imaging of mice after resection of primary tumors exhibited strong bioluminescence signals in various organs of control group mice only, suggesting distant metastasis (Fig. 6E), which was further confirmed by ex vivo imaging of resected organs (spleen, liver, and lung; Fig. 6F).

Figure 6.

IKKϵ downregulation suppresses glucose uptake, tumor growth, and metastasis in orthotopic PDAC xenografts. Luciferase-tagged control or IKKϵ-silenced MiaPaCa cells were implanted into the pancreas of athymic nude mice (n = 10 per group). A, A day prior to endpoint, mice were injected with fluorescent analogue of glucose, 2-DG (100 μL) intraperitoneally and epifluorescence measured after 24 hours using the IVIS imaging system. B, Prior to sacrificing the mice, d-luciferin (150 mg/kg body weight) was injected intraperitoneally and bioluminescence imaging data recorded using the IVIS imaging system. C,In vivo glucose uptake normalized with bioluminescent tumor measurements at the end time point and shown as relative 2-DG uptake. D and E, After sacrifice, tumors were resected and measured for weight and volume (D), and mice imaged to visualize metastases (E). F, To further confirm metastases, livers, lungs, and spleens were carefully removed from the mice and imaged separately. Representative images of mice and organs from individual groups are presented. Bars, mean ± SD (n = 10 mice); **, P ≤ 0.05.

Figure 6.

IKKϵ downregulation suppresses glucose uptake, tumor growth, and metastasis in orthotopic PDAC xenografts. Luciferase-tagged control or IKKϵ-silenced MiaPaCa cells were implanted into the pancreas of athymic nude mice (n = 10 per group). A, A day prior to endpoint, mice were injected with fluorescent analogue of glucose, 2-DG (100 μL) intraperitoneally and epifluorescence measured after 24 hours using the IVIS imaging system. B, Prior to sacrificing the mice, d-luciferin (150 mg/kg body weight) was injected intraperitoneally and bioluminescence imaging data recorded using the IVIS imaging system. C,In vivo glucose uptake normalized with bioluminescent tumor measurements at the end time point and shown as relative 2-DG uptake. D and E, After sacrifice, tumors were resected and measured for weight and volume (D), and mice imaged to visualize metastases (E). F, To further confirm metastases, livers, lungs, and spleens were carefully removed from the mice and imaged separately. Representative images of mice and organs from individual groups are presented. Bars, mean ± SD (n = 10 mice); **, P ≤ 0.05.

Close modal

PDACs are highly aggressive, exhibiting rapid growth and metastasis, which is one important reason for their high lethality in patients (2, 30, 31). As a result, efforts have focused on understanding the molecular causes of PDAC aggressiveness as well as on formulating strategies that could target this very feature of pancreatic malignancy. It is now being widely recognized that cancer cells rewire their metabolic state to fulfill the enhanced requirements of bioenergetics and biomass production, so that they can maintain their rapid proliferation (11, 13). Glucose, a major source of energy, gets metabolized through a combination of anaerobic glycolysis and oxidative phosphorylation in normal cells. However, cancer cells reprogram the glucose metabolism to support their fast growth by shifting their dependence of ATP production from oxidative phosphorylation to “aerobic” glycolysis—an observation referred to as “Warburg effect” (11, 13, 14). Moreover, altered glucose metabolism in cancer cells has also been associated with their metastatic potential and therapeutic resistance (32, 33). In these contexts, our observation of IKKϵ-mediated regulation of glucose metabolism is highly significant and suggests an important role of IKKϵ in molecular pathogenesis of PDAC. This is even more interesting considering a role of deregulated glucose metabolism in inflammation (34). In fact, chronic inflammation is known to exacerbate glucose metabolism and also promote cancer progression (35). Thus, these vicious connections of altered glucose metabolism, inflammation, and cancer, and suggested a role of IKKϵ in these phenotypes makes our findings even more noteworthy for future therapeutic and preventive intervention points.

From the mechanistic standpoint, we observed dysregulation of several genes involved in glucose metabolism upon IKKϵ silencing. This involved downregulation of genes associated with glucose and lactate transport (GLUT1, GLUT3, MCT1, and MCT4) and glycolysis (ALDOC, ENO3, GALM, GCK, HK2, HK3, etc.). As per the published data, PDAC cells are known to overexpress GLUT1, whereas the upregulation of GLUT3 along with GLUT1 has been shown in many other cancer types and correlated with poor prognosis (36–38). Similarly, expression and activity of HK2 has been observed to be upregulated in nearly all types of cancers (32). Notably, enhanced activity of HK2 is required for the initiation and maintenance of K-Ras–driven cancers, and its inhibition is shown to reduce tumor growth both in vitro and in vivo (39). There is also evidence to suggest the utility of HK2 as a prognostic marker in PDAC patients (40). The strong reliance toward aerobic glycolysis by cancer cells leads to the production of lactate by the enzyme LDHA, which is then exported to the tumor microenvironment by lactate transporters (MCT1–4) to have a multitude of functions in cancer growth and metastasis (41). Furthermore, in a majority of pancreatic tumors that express high levels of MCT4, stromal cells have also been reported to have high MCT4 expression, indicating a putative co-relation between these two events (42). Interestingly, although knockdown of IKKϵ led to the reduction of lactate efflux in the culture media, we did not observe any change in basal OCR in our study. This suggests that although IKKϵ knockdown hampers glycolytic metabolism, it does not affect oxidative phosphorylation. Furthermore, observed reduction in lactate levels could also be due to the drop in LDHA and lactate transporters, MCT1 and MCT4. This is significant as the accumulation of lactate within the cell could otherwise alter intracellular pH and subsequently induce metabolic feedback inhibitions, ultimately hampering ATP production by glycolysis (43).

Our findings also identified c-MYC as an important mediator in potentiating the effect of IKKϵ on glucose metabolism. This is very significant, as c-MYC is frequently deregulated not only in PDAC, but several other cancers as well (44, 45). c-MYC is shown to integrate cellular metabolism with survival and proliferation of cancer cells through the regulation of a number of genes (46). Under normal conditions, c-MYC transcriptional activity is controlled at several levels involving transcriptional and posttranscriptional regulation, posttranslational modification, subcellular distribution, and protein turn-over (16). Moreover, interaction of c-MYC with other nuclear proteins has also been suggested to alter its genomic occupancy and transcriptional activation. Our data demonstrate that IKKϵ does not alter mRNA levels of c-MYC in PDAC cells, but supports its nuclear retention and total protein stability. Moreover, we show that IKKϵ stabilizes c-MYC through inactivation of GSK3β, which is known to promote c-MYCT58 phosphorylation (25). Despite a previous report demonstrating direct interaction of IKKϵ with GSK3α (26), we did not observe its similar interaction with GSK3β; its phosphorylation was rather dependent on IKKϵ-mediated Akt activation. Activity of GSK3β has been demonstrated to be diminished by extracellular signals upon GSK3β-Ser-9 phosphorylation mediated by Akt and other kinases (47). Initial studies identified that constitutively activated IKKϵ could replace Myr-Akt, leading to transformation of immortalized human mammary epithelial cells (7). It was later confirmed that IKKϵ can directly phosphorylate and enhance AktT308/S473 levels even in the absence of external stimuli, and independent of other regulators (48). Our findings also demonstrated significant downregulation of Akt phosphorylation at both Thr-308 and Ser-473 sites in IKKϵ-silenced cells, which correlated with the decrease in inhibitory GSK3βS9 phosphorylation. Interestingly, despite regained Akt activation and GSK3β inactivation in IKKϵ-silenced PDAC cells, it did not lead to complete restoration of c-MYC expression, metabolic shift, and tumor growth. This may be due to technical limitation or more likely due to other IKKϵ responsive, cross-talking signaling networks that are involved in potentiating its pathobiologic functions. It is possible that IKKϵ silencing may modulate the activity of other molecular targets, such as PP2A, which are known to alter c-MYC expression and/or its activation (49, 50).

In summary, we have defined a novel role of IKKϵ in PDAC pathogenesis through its regulation of glycolytic phenotype. We have also demonstrated that IKKϵ acts as a novel regulator of c-MYC by promoting its nuclear retention and stabilization. c-MYC stability is afforded by IKKϵ-mediated Akt activation, which leads to phosphorylation-mediated inhibition of GSK3β, and, in turn, promotes nuclear retention and increase in overall transcriptional activity (Fig. 7). Our results, thus, provide strong rationale for further testing of IKKϵ as a novel molecular target to counter PDAC progression and its therapeutic management.

Figure 7.

Schematic representation of IKKϵ signaling in PDAC. IKKϵ promotes glycolytic metabolism and pancreatic tumor growth through its regulation of Akt/GSK3β/c-MYC axis. c-MYC is an important mediator of IKKϵ signaling, whose nuclear retention and stabilization is controlled by IKKϵ through a chain of events that include Akt activation, resulting in inhibitory phosphorylation of GSK3β and the escape of c-MYC from GSK3β-mediated nuclear efflux and subsequent degradation. Once stabilized, c-MYC activates multiple factors of glycolytic pathway with concomitant increase in glucose uptake and lactate efflux, leading to quick and efficient energy production to serve as prelude for synthesis of downstream molecules/factors involved in pancreatic tumor growth and metastasis.

Figure 7.

Schematic representation of IKKϵ signaling in PDAC. IKKϵ promotes glycolytic metabolism and pancreatic tumor growth through its regulation of Akt/GSK3β/c-MYC axis. c-MYC is an important mediator of IKKϵ signaling, whose nuclear retention and stabilization is controlled by IKKϵ through a chain of events that include Akt activation, resulting in inhibitory phosphorylation of GSK3β and the escape of c-MYC from GSK3β-mediated nuclear efflux and subsequent degradation. Once stabilized, c-MYC activates multiple factors of glycolytic pathway with concomitant increase in glucose uptake and lactate efflux, leading to quick and efficient energy production to serve as prelude for synthesis of downstream molecules/factors involved in pancreatic tumor growth and metastasis.

Close modal

No potential conflicts of interest were disclosed.

Conception and design: H. Zubair, S. Azim, S. Arora, A.P. Singh

Development of methodology: H. Zubair, S. Azim, S. Singh, A.P. Singh

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Zubair, S. Azim, S.K. Srivastava, M. Aslam Khan, G.K. Patel, E. Carter, S. Singh, A.P. Singh

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Zubair, S. Azim, S.K. Srivastava, A. Ahmad, A. Bhardwaj, M. Aslam Khan, G.K. Patel, J.E. Carter, S. Singh, A.P. Singh

Writing, review, and/or revision of the manuscript: H. Zubair, S. Azim, S.K. Srivastava, A. Ahmad, A. Bhardwaj, M. Aslam Khan, G.K. Patel, S. Singh, A.P. Singh

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.E. Carter, A.P. Singh

Study supervision: A.P. Singh

We would like to thank Mr. Steven McClellan, Manager, Flow Cytometry Core at the USA Mitchell Cancer Institute for his assistance with flow cytometry. We also thank Ms. Barbara Putnam (USAMCI) for careful reading of the manuscript.

This work was supported in part by NIH grants (R01CA175772 and U01CA185490 to A.P. Singh). The laboratory of S. Singh is supported by NIH grants (R01CA204801 and R03CA186223). Their laboratories also receive funding and resource support from the University of South Alabama Mitchell Cancer Institute.

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.

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