Strengthened glycolysis under hypoxia supports tumor symbiosis and hexosamine biosynthesis in pancreatic adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma is one of the most intractable and fatal cancer. The decreased blood vessel density displayed by this tumor not only favors its resistance to chemotherapy but also participates in its aggressiveness due to the consequent high degree of hypoxia. It is indeed clear that hypoxia promotes selective pressure on malignant cells that must develop adaptive metabolic responses to reach their energetic and biosynthetic demands. Here, using a well-defined mouse model of pancreatic cancer, we report that hypoxic areas from pancreatic ductal adenocarcinoma are mainly composed of epithelial cells harboring epithelial-mesenchymal transition features and expressing glycolytic markers, two characteristics associated with tumor aggressiveness. We also show that hypoxia increases the “glycolytic” switch of pancreatic cancer cells from oxydative phosphorylation to lactate production and we demonstrate that increased lactate efflux from hypoxic cancer cells favors the growth of normoxic cancer cells. In addition, we show that glutamine metabolization by hypoxic pancreatic tumor cells is necessary for their survival. Metabolized glucose and glutamine converge toward a common pathway, termed hexosamine biosynthetic pathway, which allows O-linked N-acetylglucosamine modifications of proteins. Here, we report that hypoxia increases transcription of hexosamine biosynthetic pathway genes as well as levels of O-glycosylated proteins and that O-linked N-acetylglucosaminylation of proteins is a process required for hypoxic pancreatic cancer cell survival. Our results demonstrate that hypoxia-driven metabolic adaptive processes, such as high glycolytic rate and hexosamine biosynthetic pathway activation, favor hypoxic and normoxic cancer cell survival and correlate with pancreatic ductal adenocarcinoma aggressiveness.

Ninety-five percent of patients with pancreatic cancer harbor tumors classified as pancreatic ductal adenocarcinoma (PDAC). Commonly described as a silent killer regarding its late diagnosis, PDAC is noted for its aggressiveness and its intrinsic resistance to standard chemotherapy. This specificity is probably due to a low vascular density and a prominent nontumoral cell compartment (stroma), which impact on intratumoral perfusion, therapeutic delivery, and patient outcome (1). Indeed, PDAC is characterized by numerous and severe hypoxic regions (2), a feature that has been proven to be correlated with tumor aggressiveness and poor prognosis compared with well-oxygenated tumors (3). Moreover, combined with hypoxia, the subsequent nutrient-devoid environment provides physiological selective pressure promoting expansion of the most aggressive malignant cells, particularly those acquiring mutations in genes encoding tumor suppressor protein p53 (TP53) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog protein (KRAS) (4, 5), two of the main mutations present in PDAC patients. Regarding such statements, it appears relevant to deeply explore consequences of hypoxia on PDAC carcinogenesis. In the past decade, it has been clearly highlighted that under hypoxia cancer cells develop an efficient adaptive metabolic response to ensure their survival and proliferation. Indeed, hypoxic cancer cells activate glucose uptake and glycolysis to produce pyruvate, which is then converted into lactate instead of being oxidized via the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) (6). This metabolic shift is driven by the hypoxia-inducible factor–1 (HIF1) through transcriptional activation of glycolytic genes and inhibition of those promoting OXPHOS (7). More recently, it has been shown that hypoxic cancer cells also use glutamine as a carbon fuel source for survival. After successive conversion into glutamate and α-ketoglutarate, glutamine promotes citrate synthesis and de novo lipogenesis via the isocitrate dehydrogenase–1 and –2 (IDH1 and 2)–dependent reductive carboxylation pathway (8, 9) and also supports cell proliferation through a glucose-independent TCA cycle pathway (10). Hypoxia within PDAC could then also favor a switch to both glucose- and glutamine-dependent anaerobic metabolic pathways, allowing adaptation and resistance of cancer cells to this hostile tumor microenvironment and promoting tumor development.

This metabolic switch has only been described under normoxic conditions in pancreatic cancer cell lines. Indeed, normoxic pancreatic cancer cells display high glycolytic activity and are able to metabolize glutamine (11). Furthermore, oncogenic Kras (Kras^G12D) in these cells funnels glucose into either the pentose phosphate pathway (PPP) or the hexosamine biosynthetic pathway (HBP) (12). The latter uses glucose and glutamine to generate UDP–N-acetylglucosamine (UDP–GlcNAc), a donor substrate for glycosylation reactions. Except a recent study showing a glutamine uptake following hypoxic stress in vitro and an enhanced glycolysis in hypoxic regions of PDAC (13), little effort has been invested so far to precisely determine the metabolic changes involved in hypoxia resistance and PDAC progression.

Here, we perform an exhaustive characterization of hypoxic regions in mouse PDAC and precisely define the phenotypic and metabolic features of related tumoral cells, with particular attention on glucose- and glutamine-dependent hypoxic metabolic processes.

Results

Characterization of Hypoxic Regions in PDAC.

To deepen the analysis of hypoxic regions in PDAC, we used the Pancreatic and duodenal homeobox 1-recombinase (Pdx1-Cre);Lox Stop Lox (LSL)-Kras^G12D;cyclin-dependent kinase inhibitor 2A(Ink4a/Arf)^fl/fl mouse models which develop PDAC with histological and clinical features commonly found in human PDAC (14). Importantly, as human PDAC, these tumors presented a stromal compartment favorable to the improvement of hypoxic regions. We first quantified hypoxic regions within PDAC through pimonidazole (PDZ) staining and showed that PDZ⁺ areas extended from 4.5% to 17.1% of the whole PDAC surface (Fig. 1A). As expected, these tumoral hypoxic regions were poorly vascularized since only 6.9% of CD31 staining of the whole tumor area were located in PDZ⁺ regions whereas 93.1% of CD31 staining were located outside these PDZ⁺ regions (Fig. 1B). Interestingly, although hypoxic cells were detected within ill-formed glands as well as in regions surrounding tumoral glands and necrotic zones (Fig. S1A), our histological analysis did not allow determination of whether hypoxic cells were predominantly of epithelial origin. By using wide-spectrum cytokeratin staining (epithelial marker), we demonstrated that PDZ⁺ regions contained 32.8% of epithelial cells (Fig. 1C).

Fig. 1.

Quantification and phenotypic characterization of hypoxic regions in Pdx1-Cre;LSL-Kras^G12D;Ink4a/Arf^fl/fl PDAC. (A) Individual percentage of positive hypoxic areas relative to total PDAC section (n = 6 mice) determined by immunostaining of a hypoxic marker, PDZ (scale bar, 200 µm). Immunofluorescence staining of (B) blood vessels (CD31), (C) epithelial (wide-spectrum cytokeratin, KRT, E-Cadherin) or mesenchymal (N-Cadherin) markers in PDAC hypoxic cells. Percentage of positive indicated marker within PDZ⁺ regions relative to total PDAC area is expressed as mean ± SD (n = 3 mice). M, merge (scale bar, 50 µm).

With in vitro studies, moderate hypoxia has been shown to trigger an epithelial-to-mesenchymal transition (EMT) program, characterized by the modification of known cellular markers, leading to increased aggressiveness of many human neoplasic cell lines (15). Here, we noted that hypoxic cells lost E-cadherin expression to the benefit of N-Cadherin expression (mesenchymal marker) given that the percentage of E-Cadherin staining in PDZ⁺ regions was 24.4% and that of N-Cadherin was 36.3% (Fig. 1C). These in vivo data show that epithelial cells, representing a significant pool of cells in hypoxic regions of PDAC, enter an EMT program attesting of their potential aggressiveness.

Hypoxia Strongly Participates in the Glycolytic Phenotype of PDAC.

It has been shown that increased tumor aggressiveness is correlated with glycolytic phenotype of carcinomas (6). In solid tumors, hypoxia promotes the shift from OXPHOS to glycolytic mode, leading to increased glucose capture and lactic acid production by tumor cells (anaerobic “glycolysis”), a process also called the Warburg effect when detected in well-oxygenated tumor cells (aerobic “glycolysis”). Here, we aimed to gain further insight into glycolytic activity of hypoxic pancreatic cancer cells in vivo as an index of their potential aggressiveness and to compare this activity to the one of normoxic cells. We first investigated the expression of genes involved in glycolytic metabolism such as Hexokinase 2 (Hk2), Phosphoglycerokinase 1 (Pgk1), Pyruvate dehydrogenase kinase, isozyme 1 (Pdk1), and Lactate dehydrogenase a (Ldha) as well as glucose and lactate transporters such as Glucose transporter 1 (Glut1) and Monocarboxylate transporter 4 (Mct4). As suspected, all of these genes were strongly increased in tumors versus control pancreata (Fig. 2A and Fig. S1B), meaning that PDAC is a highly glycolytic tumor, able to produce and extrude lactate into extracellular space. Moreover, 50.2% and 58.2% of GLUT1 and MCT4 staining were located in PDZ⁺ regions, respectively (Fig. 2B), suggesting that in PDAC a major part of glycolytic activity takes place in hypoxic areas. To more precisely characterize the glycolytic activity of pancreatic cancer cells submitted to hypoxia, we used an in vitro epithelial cancer cell model (PK4A cells) issued from Pdx1-Cre;LSL-Kras^G12D;Ink4a/Arf^fl/fl PDAC (Fig. S1C). Under 1% O₂, a low enough level of hypoxia able to stabilize HIF1α (Fig. S1D), the transcription of genes encoding for glycolytic enzymes and glucose and lactate transporters was up-regulated (two- to sevenfold) compared to the one of normoxic PK4A cells (Fig. 2C and Fig. S1E). We also found that these hypoxic cells were highly metabolically active, as after 48 and 72 h of hypoxia, they consumed and released twofold more glucose and lactate, respectively, than normoxic cells (Fig. 2D). These results suggest that PDAC-hypoxic areas containing highly glycolytic cells represent niches of extremely aggressive cells widespread in the tumor mass.

Fig. 2.

Exacerbated increase of glycolysis, glucose uptake, and lactate release in PDAC and PK4A cells under hypoxia. (A) Hk2, Glut1, and Mct4 mRNA levels in LSL-Kras^G12D;Ink4a/Arf^fl/fl control pancreata (n = 3 mice) and PDAC (n = 3 mice) normalized to 36B4 mRNA levels. P value is relative to the first control pancreas expression. (B) Immunofluorescence staining of GLUT1 and MCT4 transporters in PDZ⁺ regions of PDAC. Percentage of GLUT1⁺ or MCT4⁺ areas in hypoxic regions is indicated as in Fig.1B (scale bar, 50 µm). (C) Hk2, Glut1, and Mct4 mRNA levels in PK4A cells cultured during 24, 48, and 72 h in hypoxia (1% O₂) or normoxia and normalized as in A. P value is relative to respective normoxic value. (D) Glucose (Upper Left) and lactate concentration (Lower Left) assessed in PK4A supernatant every 24 h over a 72 h period of normoxia (N) or hypoxia (H) and normalized to respective viable cell number. P value is relative to respective normoxic value. (Right) Fold increases in glucose uptake (Glc) and lactate production (Lact) in hypoxic PK4A cells relative to corresponding normoxic values are illustrated. P values (all < 0.05) are relative to glucose uptake (a) or lactate production (b) measured after 24 h under hypoxia. For all figures, M, merge. *P < 0.05 and **P < 0.001. Error bars indicated SD (shown results are representative of at least three independent experiments).

Lactate from Glycolytic Hypoxic Cells Fuels Growth of Normoxic Cells.

Previous reports have shown that oxygenated cancer cells can metabolize lactate through the oxidative pathway to satisfy their energetic and biomass needs (metabolic symbiosis) (16, 17). We therefore speculated that a lactate exchange between glycolytic hypoxic cells and neighboring normoxic–oxidative cells could exist in mouse PDAC. We observed that mRNA levels of Mct1 (the monocarboxylate transporter 1 responsible for lactate uptake by normoxic cells) were four- to 17-fold higher in mouse PDAC compared with control pancreata (Fig. 3A). Moreover, MCT1 staining was highly increased in normoxic areas, with 65% of the staining localized in PDZ negative areas (Fig. 3B). To reproduce these observations in human tissue using in vivo settings, we generated patient-derived pancreatic tumor xenografts that recapitulate the anatomopathologic characteristics of human PDAC (Fig. S2A) and in which hypoxic areas can be stained using PDZ (Fig. S2B). In this model, MCT1 was mainly localized in cells close to a strong hypoxic area in which its expression was notably lessened (Fig. 3C), whereas MCT4 staining strongly matched with hypoxic regions as already shown in Fig. 2B. Detection of MCTs in resected human pancreatic tumors revealed the presence of both transporters in plasma membrane of epithelial cells constituting tumoral glands. However, only MCT4 was present in epithelial cells disseminated in the stroma, meaning that the two transporters also differ in their localization in the clinical context (Fig. S2C).

Fig. 3.

Lactate is used as an alternative carbon source by normoxic PK4A cells. (A) Mct1 mRNA levels in control pancreata and PDAC. Data and P value are expressed as in Fig. 2A. (B) Immunofluorescence costaining of MCT1 with PDZ in mouse PDAC. Percentage of MCT1⁺ area within normoxic regions surrounding PDZ⁺ cells is indicated and expressed as in Fig. 1B. (C) Immunofluorescence costaining of MCT1 and MCT4 with PDZ in patient-derived pancreatic tumor xenografts (scale bar, 50 µm). (D) Mct1 mRNA levels in PK4A cells cultured and expressed as in Fig. 2C. (E) Lactate concentration in supernatant from PK4A cells cultured in reduced serum media supplemented or not with sodium L-lactate. Values are normalized to corresponding viable cell numbers. P value (all < 0.05) is relative to 24 h lactate concentration measured in each media (a and b, respectively). (F) Number of PK4A cells cultured as in E. P value is relative to cell numbers measured in corresponding lactate-free media.

Finally, we confirmed on PK4A cells that Mct1 transcripts were up-regulated in normoxic conditions and that hypoxia impeded this time course induction (Fig. 3D). Interestingly, PK4A cells cultured under normoxia in lactate-supplemented media consumed up to 50% of this added lactate over a 3 d period (Fig. 3E), whereas PK4A cells cultured without lactate in media synthesized and released lactate as previously shown (Fig. 2D, Lower Left). This increased lactate consumption promoted PK4A proliferation rate with a similar time course (Fig. 3F). We observed a comparable proliferative advantage when PK4A cells were cultured under normoxia with lactate-enriched conditioned medium from hypoxic cells (Fig. S2D). Therefore, besides showing that normoxic cells are less glycolytic than hypoxic ones (Fig. 2), our data further document that pancreatic normoxic cancer cells are metabolically flexible and are able to use lactate produced by hypoxic cells as an alternative substrate to proliferate.

Glutamine Is Necessary for Hypoxic Pancreatic Tumor Cell Survival.

Although hypoxic PDAC cells are highly glycolytic with important glucose consumption, it appears necessary to evaluate their ability to metabolize glutamine, which is the second most important source of carbon after glucose and the first supplier of nitrogen. To measure the glutamine demand of pancreatic tumor cells in our in vitro and in vivo pancreatic cancer models when submitted to hypoxic stress, we first determined the expression of key enzymes, glutaminase (GLS) and GLS2, which catalyze glutamate production from glutamine. In PK4A cells, Gls2 displayed higher levels of expression than Gls (2.5- vs. 1.5-fold increase) following 48 h of 1% O₂ treatment (Fig. 4A), and costaining of GLS2 and PDZ on mouse PDAC sections revealed that 50% of GLS2 staining is localized in PDZ⁺ areas (Fig. 4B). We next assessed glutamine utilization in PK4A cells by measuring intracellular glutamate production when they were cultured in media with or without glutamine. As expected, we observed that hypoxic PK4A cells were able to produce glutamate and that glutamine accounted for the major source of this glutamate production (Fig. 4C). Moreover, we showed that hypoxic cells cultured in glutamine-deficient medium lost their ability to maintain viability (Fig. 4D), as well as their clonogenic growth capacity (Fig. 4E). Thus, viability maintenance, proliferation, and colony formation capacities of hypoxic pancreatic tumor cells appear strongly dependent on glutamine consumption.

Fig. 4.

Glutaminolysis is required for proliferation of pancreatic tumoral cells in hypoxia. (A) Gls and Gls2 mRNA levels in PK4A cells cultured during 15, 24, and 48 h in hypoxia (1% O₂). Data are normalized as in Fig. 2A, and P value is relative to respective Gls expression. (B) Immunofluorescence staining of GLS2 in PDZ⁺ PDAC regions. Percentage of GLS2⁺ areas in hypoxic regions is indicated and expressed as in Fig. 1B. (C) Intracellular glutamate levels in PK4A cells cultured during 24 h in hypoxia, as in A, in complete, glutamine-free media, supplemented or not with 4 mM of glutamine (+Gln and −Gln, respectively). Glutamate was normalized to corresponding viable cell numbers, and P value is relative to respective glutamate levels measured in complete media. (D) Viable PK4A cell numbers (Left) and bright-field images of these cells (Right) cultured during 48 h in hypoxia as in C. P value is relative to corresponding cell numbers measured in Gln-added media (scale bar, 200 µm). (E) Representative clonogenic assay performed with PK4A cells cultured in hypoxia as in A during 5 d in reported media.

HBP Is Promoted in Hypoxic Pancreatic Tumor Cells.

Recent data revealed that cancer cells are able to coordinate glucose and glutamine metabolisms through the HBP pathway (Fig. S3A) (18). As we have shown that hypoxic pancreatic cancer cells could metabolize glucose as well as glutamine, it is therefore conceivable that the HBP pathway can be activated under hypoxic condition in pancreatic cancer cells. Among genes encoding key enzymes involved in nucleotide sugar UDP–GlcNAc synthesis generated after HBP pathway activation (Fig. S3A), glutamine fructose-6-phosphate amidotransferase 1 (Gfpt1) was 1.5-fold up-regulated, whereas Gfpt2 was increased up to ninefold in PK4A cells following 15 h of hypoxic treatment (Fig. 5A). UDP–GlcNAc is the limiting substrate for the O-linked GlcNAc posttranslational modifications (PTMs) (O-GlcNAcylation) of proteins (Fig. S3A), and interestingly, O-GlcNAc transferase (Ogt) and O-GlcNAcase (Oga) transcripts, encoding O-GlcNAc cycle enzymes, were also moderately up-regulated in hypoxic PK4A cells (Fig. S3B). This enhanced expression of Gfpts, Ogt, and Oga was also found in vivo within mouse PDAC compared to control pancreata (Fig. 5B and Fig. S3C). At the protein level, in vivo immunodetection of GFPT2 in mouse PDAC also revealed a strong expression of this enzyme within epithelial cells localized in tumor glands or disseminated in the stromal compartment (Fig. 5C). Moreover, all PDZ⁺ cancer cells strongly expressed GFPT2 as shown in PDAC serial sections (Fig. 5C). Regarding PTMs, we observed a 40% increase of O-GlcNAc protein levels in hypoxic PK4A cells compared with normoxic cells (Fig. 5D). Interestingly, when PK4A cells were cultured in glucose-free media, addition of GlcNAc metabolite increased the total amount of O-GlcNAc protein from 86% (normoxia) to 150% (hypoxia). Finally, we observed that such O-GlcNAcylation processes were drastically decreased in glucose-free media or after glutamine starvation induced by addition of unmetabolized glutamine analog (6-diazo-5-oxo-L-norleucine, DON ) in hypoxic conditions relative to normoxic ones [43% vs. 30% for glucose (Glc)⁻ and 64% vs. 50% for glutamine (Gln)⁻]. To correlate those data with cell behavior, we investigated whether inhibition of O-GlcNAcylation of proteins could affect hypoxic PK4A cell viability. As suspected we observed a significant decrease in hypoxic cell number following inhibition of HBP rate-limiting enzymes (GFPTs) by azaserine (Fig. 5E). Taken together, our results provide evidence that activation of the HBP pathway is necessary for hypoxic pancreatic cancer cells to survive.

Fig. 5.

Activation of the HBP in hypoxic pancreatic cancer cells. (A) Gfpt1 and Gfpt2 mRNA levels in PK4A cells cultured during 8, 15, and 24 h in hypoxia (1% O₂). Data are expressed as in Fig. 4A. P value is relative to respective 8 h expression. (B) Gfpt1 and Gfpt2 mRNA level in PDAC and control pancreata. Data are expressed as in Fig. 2A. (C) GFPT2 and PDZ staining of serial mouse PDAC sections. Hypoxic regions are delineated by red line, and GFPT2⁺ cells (►) in tumor glands or disseminated in stroma are indicated (scale bar, 50 µm). (D) O-GlcNAc proteins expression in PK4A cells cultured during 24 h in normoxia or hypoxia in complete media supplemented or not with DON (30 µM) and in glucose-free media supplemented or not with GlcNAc (15 mM). O-GlcNAc–β-actin protein ratios are expressed relative to complete media ratio measured in normoxia and are indicated below immunoblots. (E) Number of viable PK4A cells cultured during 48 h in hypoxia in complete media supplemented or not with azaserine (10 µM). P value is relative to corresponding viable cell numbers determined in complete media.

Discussion

PDAC is one of the most hypoxic cancer together with the more unfavorable patient prognosis, as mortality rate is almost equivalent to its incidence (2, 19). In PDAC, hypoxic cancer cells, known to be resistant to chemotherapies, probably constitute cell niches that locally participate to disease progression and recurrence (20). Thereby, deciphering the metabolic pathways that contribute to the resistance and proliferation of these hypoxic niches could highlight new metabolic targets to limit malignant progression of this cancer.

In this report, we perform a quantitative and qualitative analysis of hypoxic regions within pancreatic tumors. We show that PDAC exhibits a considerable number of hypoxic areas, which certainly depends on the advanced stage of the disease. We also determine that one-third of hypoxic regions is composed of epithelial cells. Moreover, these epithelial cells present EMT features, suggesting that, in PDAC, hypoxia may induce a reprogramming of epithelial cancer cells toward a mesenchymal invasive phenotype, as previously suggested in vitro (15). Surprisingly, acquisition of the N-Cadherin mesenchymal marker under hypoxic condition is nutrient-dependent as glucose and glutamine deprivations prevent it (Fig. S4). This suggests that glycolytic and glutaminolytic activity of epithelial cells is tightly linked to their EMT program. Although glycolytic activation process occurring during PDAC progression has been extensively described in oxygenated cells, the relevance of such metabolic pathway activation in anaerobic conditions remained unclear. Our data provide evidence that pancreatic hypoxic cells compared with cells exhibiting aerobic glycolysis (Warburg effect) display a higher glycolytic potential, resulting in a stronger activation of all enzymes and transporters involved in glucose uptake and lactic acid formation. Interestingly, it is reported that exacerbated glycolytic activity correlates with a more aggressive phenotype in breast cancer cells (21), and that acidosis due to increased lactic acid in tumor microenvironment is indirectly associated to the extracellular matrix breakdown, a mechanism that favors invasiveness (6). Hence, in the context of PDAC, we suggest that hypoxia, by promoting both glycolytic activity (and associated lactate efflux) and invasive phenotype of pancreatic epithelial cells, is mainly responsible for the tumor aggressiveness.

It is now well established that excess of lactate released by highly glycolytic cells, besides promoting tumor invasion, can also be used as a glucose-alternative carbon source by neighboring oxygenated cancer cells, as it has been recently described in colon cancer cell lines (16). Here, we provide evidence that such tumoral symbiosis between hypoxic and normoxic cancer cells also exists in pancreatic cancer, as lactate present in culture media is taken up by oxygenated pancreatic cells that use it as a fuel source to increase their proliferation rate. These results are reinforced by the fact that, in mouse PDAC, expression of MCT1, which is involved in the uptake of lactate, is switched on in normoxic cancer cells and reduced in hypoxic ones. The same pattern of expression of MCT1 is observed in patient-derived xenografts, suggesting that such tumoral symbiosis may also exist in human PDAC. All together, these data lead to the conclusion that lactate constitutes a crucial energetic metabolite allowing fuel exchanges between hypoxic and normoxic compartments in the pancreatic tumor. A recent study, showing that alteration of MCT1 and MCT4 membrane trafficking by CD147 inhibition reduces the proliferation rate of aerobic glycolytic pancreatic cancer cells (22), strengthens the fact that the export of lactate by hypoxic cells and its uptake by normoxic ones are essential for the malignant phenotype of PDAC.

A role of oncogenic KRAS in reprogramming cancer cell metabolism through activation of glucose and glutamine metabolism and their derived pentose phosphate (PPP) and hexosamine pathways has been documented in various cancer cell lines including pancreatic ones (12, 23). Once taken up by cancer cells, glutamine is converted into glutamate partly by GLS enzymes through the glutaminolysis pathway. In pancreatic cancer cells, the isoform 2 (GLS2) is preferentially expressed in hypoxic areas, suggesting that glutamate production in hypoxic cells is essentially dependent of GLS2 and not GLS. Furthermore, hypoxic pancreatic cancer cells, in addition to their glutamine-dependent cell growth and their high glucose consumption, activate the HBP pathway mainly through the rate-limiting enzyme GFPT2, which is the most activated GFPT enzyme under hypoxia. Finally, the increase of O-GlcNAc protein levels under hypoxia compared with normoxia is clearly dependent on both glucose and glutamine availability and it sustains tumor cell viability. O-GlcNAcylation is a PTM that stabilizes numerous factors involved in tumorigenic processes such as myc myelocytomatosis viral oncogene homolog protein (c-Myc), TP53, and β-catenin (24, 25). It also participates in the phenotype of cancer cells by promoting aneuploidy and by activating key oncogenic signaling pathways including insulin, fibroblast growth factors, and transforming growth factor–β activated pathways (26). Here, in the context of PDAC, we suggest that GlcNAcylation allows cells to promptly react to microenvironmental stress situations such as hypoxia through stabilization of factors yet to be determined and involved in resistance to hypoxia and in its associated metabolic pathways. Very recently, Yi et al. showed that phosphofructokinase 1, a key enzyme of glycolysis flux, is O-GlcNAcylated and that this PTM responsible for its inactivation redirects glucose flux through pathways critically involved in tumor growth as PPP (27). Therefore, the crucial role of HBP activation to maintain cell viability under hypoxic conditions might involve regulation of mediators including glycolytic enzymes.

Taken together, our results suggest that enhanced glucose metabolism in hypoxic pancreatic cancer cells represents a key regulating pathway, as its end-product, lactate, is required for growth of neighboring normoxic cells, and its by-products cooperate with glutamine to enable PTMs, all favoring PDAC progression (Fig. 6). These results shed light on crucial metabolic pathways/enzymes activated in PDAC hypoxic regions that are considered as adaptive landscapes for selection of aggressive and invasive cells. Therefore, targeting of those pathways could eliminate hypoxic niches and thus limit PDAC progression and associated metastases.

Fig. 6.

Schematic representation of glucose- and glutamine-derived metabolic pathways activated by hypoxia in pancreatic cancer cells. Hypoxia enables PDAC progression through (i) the increased glucose flux and subsequent glycolysis and lactate production [lactate, released in extracellular space, increases invasive potency of tumoral cells through acidification of pericellular pH (acidosis) and favors growth of normoxic cells that use it as a fuel source]; (ii) the increased glutaminolysis, which provides cells with glutamate that once converted into a TCA intermediate metabolite, promotes biomass production; and (iii) the HBP that requires both major extracellular carbon sources, glucose and glutamine, to enable O-GlcNAC modifications of protumoral proteins. Activation of these various metabolic pathways by hypoxia contributes to PDAC progression.

Materials and Methods

Cells and Reagents.

Cell culture media and conditions are described in SI Materials and Methods. Hypoxia was performed at 1% (vol/vol) O₂ and 5% (vol/vol) CO₂ in nitrogen atmosphere.

Mouse Strains and Tissue Collection.

Pdx1-Cre;Ink4a/Arf^fl/fl;LSL-Kras^G12D mice were obtained by crossing the following strains: Pdx1-Cre;Ink4a/Arf^fl/fl and LSL-Kras^G12D mice kindly provided by Dr. D. Melton (Harvard Stem Cell Institute, Cambridge, MA), Dr. R. Depinho (Dana-Farber Cancer Institute, Boston) and Dr. T Jacks (David H. Koch Institute for Integrative Cancer Research, Cambridge, MA), respectively. PDAC-bearing 8–12-wk-old male mice were killed with their mating control littermates. Pieces of tumor or control pancreata were fixed in 4% (wt/vol) formaldehyde or frozen in cold isopentane for further immunostaining analysis or directly homogenized in 4 M guanidinium isothiocyanate lysis buffer for efficient pancreatic RNA extraction according to Chirgwin’s procedure (28). Immunodetection of cellular hypoxia required an i.p. injection of PDZ hydrochloride (60 mg/kg; Hypoxyprobe) 4 h before sacrifice. All animal care and experimental procedures were performed in agreement with the Animal Ethics Committee of Marseille.

Xenografts.

Patient-derived pancreatic tumor pieces (1 mm³) were embedded in Matrigel before to be s.c. implanted into flank of adult male Swiss nude mice (Charles River Laboratories) under isoflurane anesthesia (induction, 4% (vol/vol); maintenance, 1.5% (vol/vol)). Tumors were measured weekly with a caliper until tumor volume reached 1 mm³. At 4 h after intratumoral injection of PDZ hydrochloride, pieces of tumor were removed, fixed in 4% (wt/vol) formaldehyde, or frozen in cold isopentane for further analysis.

Immunohistochemistry, Immunofluorescence, Western Blots, and Primary Antibodies.

Protocols and antibodies used for immuno-detection of proteins in tissue sections or in cell protein lysates are described in SI Materials and Methods.

Image Acquisition and Analysis.

Quantification of tissue sections stained for PDZ as well as various markers was determined as in SI Materials and Methods using ImageJ software (National Institutes of Health).

Real-Time PCR.

RT-PCR was performed as in SI Materials and Methods. Primers used are shown in Table S1.

Metabolites Assays.

Measurements of glucose, lactate, and glutamate were performed by either Yelen (Marseille, France) or with a Biovision kit, as described in SI Materials and Methods.

Statistical Analysis.

Comparisons between experimental groups were performed either by Student t test or by ANOVA or MANOVA, with subsequent post hoc test using SAS statistical software. P < 0.05 was considered statistically significant.