HuR is a post-transcriptional regulator of core metabolic enzymes in pancreatic cancer

Abstract

Cancer cell metabolism differs from normal cells, yet the regulatory mechanisms responsible for these differences are incompletely understood, particularly in response to acute changes in the tumor microenvironment. HuR, an RNA-binding protein, acts under acute stress to regulate core signaling pathways in cancer through post-transcriptional regulation of mRNA targets. We demonstrate that HuR regulates the metabolic phenotype in pancreatic cancer cells and is critical for survival under acute glucose deprivation. Using three pancreatic cancer cell line models, HuR-proficient cells demonstrated superior survival under glucose deprivation when compared with isogenic cells with siRNA-silencing of HuR expression (HuR-deficient cells). We found that HuR-proficient cells utilized less glucose, but produced greater lactate, as compared with HuR-deficient cells. Acute glucose deprivation was found to act as a potent stimulus for HuR translocation from the nucleus to the cytoplasm, where HuR stabilizes its mRNA targets. We performed a gene expression array on ribonucleoprotein-immunoprecipitated mRNAs bound to HuR and identified 11 novel HuR target transcripts that encode enzymes central to glucose metabolism. Three (GPI, PRPS2 and IDH1) were selected for validation studies, and confirmed as bona fide HuR targets. These findings establish HuR as a critical regulator of pancreatic cancer cell metabolism and survival under acute glucose deprivation. Further explorations into HuR's role in cancer cell metabolism should uncover novel therapeutic targets that are critical for cancer cell survival in a metabolically compromised tumor microenvironment.

Keywords: HuR; cancer metabolism; pancreatic cancer; post-transcriptional gene regulation.

Figure 1. HuR protects pancreatic cancer cells against glucose-deprivation. In bar graphs: siCTRL, black bars; siHuR, gray bars (*p < 0.05). In survival curves (unless indicated): siCTRL are solid lines; siHuR are dashed lines. (A) Representative qPCR 24 h after transfection in MiaPaCa2 cells. (B) Representative immunoblot 48 h after transfection in MiaPaCa2 cells. (C) Annexin V staining in MiaPaCa2 cells. (D) Trypan blue staining in MiaPaCa2 cells. (E) PicoGreen assays at 7 d in MiaPaCa2 cells. (F) Three-week soft-agar colony formation assays in MiaPaCa2 cells. PicoGreen assays at seven days in (G) BXPC3 and (G) PANC1 cells. (I) Representative immunoblot 72 h after transfection in PANC1 cells. (J) PicoGreen assay at seven days in PANC1.HuR (double line-top) vs. PANC1.EV (single line-bottom).

Figure 2. HuR protein is activated to the cytoplasm under glucose deprivation. (A) Immunoblots of whole cell (left) and cytoplasmic (right) lysates from MiaPaCa2 cells cultured for 6 h in media with the indicated glucose concentrations. Gemcitabine (GEM) (1 µM) was used as a positive control for cytoplasmic HuR activation. HuR (and GEM) levels are quantified at each glucose concentration and numeric values are displayed normalized to control glucose conditions below the corresponding cytoplasmic HuR band. Immunofluorescence at 12 h (B) and immunoblots at 6 h (C) of BxPC3 cells in normal DMEM (25 mM glucose), 1 mM glucose DMEM and 1 µM gemcitabine (positive control). Nuclei are counterstained in blue (DAPI). Immunoblots of (D) PANC1 cells after incubation in the indicated glucose concentrations for 6 h. Relative HuR levels are quantified, as described for panel (A).

Figure 3. HuR expression affects lactate production of pancreatic cancer cells. Lactate levels in the media over time. siCTRL are solid lines; siHuR are dashed lines. The starting glucose concentration for these experiments was 5 mM. (A) MiaPaCa2 cells, (B) BXPC3 cells and (C) PANC1 cells. Lactate levels are normalized to total cell count in each well.

Figure 4. HuR expression affects glucose uptake into pancreatic cancer cells independent of GLUT1. In glucose concentration curves: siCTRL are solid lines; siHuR are dashed lines. (A) Glucose levels in the media (5 mM starting glucose) over time in MiaPaCa2 cell culture. (B) Box plot of intracellular glucose levels detected by GC/MS and LC/MS/MS platforms in MiaPaCa2 cells incubated in 25 mM glucose (*p < 0.1). Glucose levels in the media (5 mM starting glucose) over time in (C) BxPC3 cells and (D) PANC1 cells. (E) Ribonucleotide immunoprecipitation and qPCR for GLUT1 demonstrating that GLUT1 does not bind HuR (gray bars), relative to IgG (black bars) in MiaPaCa2 and PANC1 cell lines. BxPC3 cells demonstrated mild enrichment of GLUT1 mRNA in the HuR sample relative to the IgG sample. The HuR target, dCK, served as a positive control (data not shown, MiaPaCa2: 8 fold increased binding; PANC1: 4 fold increased binding; BxPC3: 3-fold increased binding). (F) qPCR of GLUT1 mRNA in MiaPaCa2.siHuR (gray bars) and MiaPaCa2.siCTRL cells (black bars). (G) Immunoblots for HuR, GLUT1 and α tubulin of whole cell MiaPaCa2 lysates 72 h following transfection with siRNA oligs. (H) Flow cytometric analysis of GLUT1 cell surface expression of MiaPaCa2.siHuR (blue) and MiaPaCa2.siCTRL cells (red, set to a value of 1 in the bar graph).

Figure 5. Identification of HuR metabolic target mRNAs through ribonucleotide immunoprecipitation assays. mRNA enriched by HuR immunoprecipitation were compared with an IgG control sample in MiaPaCa2 cells with an 84-gene glucose metabolism qPCR Array. (A) Heatmap/clustergram: red represents increased transcript levels with HuR RNP-IP compared with control, and green represents decreased levels. Genes enriched in the HuR sample at levels 4-fold or greater than the IgG sample are highlighted with a purple bar to the left of the heat map. (B) Eleven HuR targets are presented and grouped by pathway. RNA levels represent fold-change enrichment to HuR, relative to IgG. Data are normalized to GAPDH and the red dashed line (also present in panels C and D) indicate the predetermined cutoff used to define highly enriched HuR targets (greater than 4-fold enrichment in the HuR sample over IgG control). IDH1 was grouped with TCA cycle enzymes for the purposes of presentation, but technically is an isoenzyme of mitochondrial IDH and resides in the cytosol. GPI, IDH1 and PRPS2 were validated as HuR-bound mRNA targets by HuR RNP-IP and q-PCR in (C) BxPC3 and (D) PANC1 cell lines. HuR RNP-IP (gray bars) and IgG RNP-IP (black bars). PCR Arrays were normalized to GAPDH. qPCR assays were normalized to 18S.

Figure 6. HuR silencing alters expression of metabolic targets. (A) Gene target mRNA expression 48 h after transfection (normalized to 18S) in MiaPaCa2.siCTRL (black bars) and MiaPaCa2.siHuR cells (gray bars). Whole cell protein lysates were assayed for metabolic proteins at 72 h in (B) MiaPaCa2, (C) BxPC3 and (D) PANC1 cells. (E) MiaPaCa2 cells were incubated in 1 mM glucose for the indicated time points and metabolic target protein expression was determined. (F) Protein expression of HuR targets over time from panel (E) are quantified and graphed. MiaPaCa2.siCTRL are solid lines; MiaPaCa2.siHuR are dashed lines. * highlights the later time points in panel (E) with increased target expression in MiaPaca.siCTRL cells.