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.

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).

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).

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.

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.

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.

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.