Tumour hypoxia induces a metabolic shift causing acidosis: a common feature in cancer

Abstract

Maintenance of cellular pH homeostasis is fundamental to life. A number of key intracellular pH (pHi) regulating systems including the Na(+)/H(+) exchangers, the proton pump, the monocarboxylate transporters, the HCO(3)(-) transporters and exchangers and the membrane-associated and cytosolic carbonic anhydrases cooperate in maintaining a pHi that is permissive for cell survival. A common feature of tumours is acidosis caused by hypoxia (low oxygen tension). In addition to oncogene activation and transformation, hypoxia is responsible for inducing acidosis through a shift in cellular metabolism that generates a high acid load in the tumour microenvironment. However, hypoxia and oncogene activation also allow cells to adapt to the potentially toxic effects of an excess in acidosis. Hypoxia does so by inducing the activity of a transcription factor the hypoxia-inducible factor (HIF), and particularly HIF-1, that in turn enhances the expression of a number of pHi-regulating systems that cope with acidosis. In this review, we will focus on the characterization and function of some of the hypoxia-inducible pH-regulating systems and their induction by hypoxic stress. It is essential to understand the fundamentals of pH regulation to meet the challenge consisting in targeting tumour metabolism and acidosis as an anti-tumour approach. We will summarize strategies that take advantage of intracellular and extracellular pH regulation to target the primary tumour and metastatic growth, and to turn around resistance to chemotherapy and radiotherapy.

Fig 1

pHi recovery after an acid-load (ammonium pre-pulse) in CCL39 wild-type (CCL39-wt) or mutant cells lacking NHE-1 (CCL39-nhe-1⁻). Exponentially growing non-neoplastic Chinese hamster lung fibroblast CCL39 cells seeded on glass cover slips were incubated for 30 min. in glucose/saline solution bicarbonate-free, HEPES-buffered solution adjusted to pHo 7.4. The pH-sensitive fluorescent dye 2′,7′ bis (carboxyethyl)-5-(6)-carboxyfluorescein (BCECF-AM) was then added to the cells for 5 min. before transfer of cells to a laminar flow cell chamber perfused with the same solution. Cells were maintained for 10 sec. and then a NH₄Cl solution (NH₄⁺) that causes alkalinization (ammonium pre-pulse) was added. The cells were shifted to a pHo 7.4 Na⁺/NH₄Cl-free solution (0 mM Na⁺) (containing choline chloride instead of NaCl), which causes intracellular acidification due to extrusion of NH₃ while preventing exchanger activity. Re-introduction of Na⁺ (120 mM Na⁺) allowed for exchanger activity. Ratiometric measurement of the fluorescence of 50 randomly selected individual cells per cover slip was performed in a workstation (Acquacosmos). The pHi was estimated by in situ two-point calibration (pHo 6.6 to 7.6) with perfusion of glucose/saline KCl/nigericin containing solutions to measure the pHi as a function of the fluorescence ratio.

Fig 2

Role of NHE-1 in the control of energy metabolism, pH regulation and nude mice tumorigenicity. Tumour incidence in nude mice of the hamster lung cell lines derived from CCL39 cells. Parental hamster lung fibroblasts (wt), mutant cells impaired in respiration (res⁻), mutant cells impaired in glycolysis due to a lack of phosphoglucose isomerase activity (gly⁻), mutant cells lacking NHE-1 activity (nhe-1⁻) and mutant cells lacking both functions res⁻ nhe-1⁻ and gly⁻nhe-1⁻, were transformed with the H-ras oncogene and inoculated (1 × 10⁶ cells/mice) by subcutaneous injection into athymic nude mice. The combination of a defect in pHi regulation through mutation of nhe-1 (nhe-1⁻) or of high glycolysis (res⁻) resulted in transient tumour take (percentage of tumour bearing mice represented by dotted lines) but which regressed substantially. The full line shows the final tumour take. Combination of exacerbated glycolysis and a defect in pHi regulation through nhe-1 mutation (res⁻ nhe-1⁻) resulted rarely in tumour take.

Fig 3

HIF-1 mediates microenvironmental changes and cellular adaptation to tumour hypoxia. After translocation to the nucleus, dimerization of HIF-1α with the constitutive HIF-1β, forms an active transcription factor that only exists in a low oxygen tension (hypoxia). Heterodimerization and DNA binding involve interaction between the N-terminal basic-helix-loop-helix (bHLH) and PerArntSim domains. HIF-1 binds hypoxia responsive elements (HRE) in target genes and activates the transcription of genes enhancing adaptation and cell survival in a hypoxic environment. These target genes play key roles in oxygen sensing via the prolyl hydroxylases 2 and 3 (phd2, phd3), in decreased mitochondrial respiration through the induction of pdk1, in erythropoiesis (epo), in angiogeneisis via the expression of vascular endothelial growth factor (vegf), angiopoietin 2 (ang2), interleukin 8 (il8), in increased glycolytic enzymes as for example glucose transporters (glut-1), lactate dehydrogenase-a (ldh-a), in pHi and pHo regulation with HIF-1 induction of the carbonic anhydrase 9 and 12 (ca9, ca12) and the mct4, in autophagy triggered by Bcl-2/adenovirus EIB 19-kD interacting protein 3 (bnip3) and bnip3l, and in migration/invasion mediated by HIF induction of matrix metalloproteinase 2 (mmp2).

Fig 4

Diagram of the glycolytic pathway. Glycolysis produces two ATP molecules from one glucose molecule consumed. The expression of many of the genes coding for enzymes of the pathway are HIF-1-induced (and oncogenes-induced); including HK1/2, phosphoglucose isomerase, PFK, aldolase, PGK1, phosphoglycerate mutase, enolase, pyruvate kinase (M2-PK) and the lactate dehydrogenase-a (LDH-A). NAD⁺ recycling is of capital importance for ATP generation. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH).

Fig 5

The carbonic anhydrase catalytic reaction, localization and domain structure CAs catalyse the reversible hydration of CO₂ to H⁺ and HCO₃⁻. CAIX and CAXII are cell membrane located, as demonstrated by immunofluorescence of CAIX-expressing cells, in contrast to the cytosolic distribution of CAII. Domain organization of the membrane-bound hypoxia inducible CAIX and CAXII, and the constitutive cytosolic CAII: PG = proteoglycan-like domain, CA = catalytic domain, TM = transmembrane domain, IC = intracellular C-terminal tail.

Fig 6

Immunohistochemical analysis of HIF-1α and CAIX expression in mice xenograft tumours. Tumour sections obtained from subcutaneous injection of colon carcinoma LS174Tr cells were stained with an anti-CAIX monocalonal antibody M75 and showed correlation between CAIX expression and regions of HIF-1α⁺ staining. A relationship between CAIX localization and regions of hypoxia and necrosis, was also noted. Staining of CAIX is predominantly membranous while HIF-1α is localized in the nucleus. Magnification 20×.

Fig 7

Up-regulation of glycolysis and pHi regulation: hallmarks of invasive cancers. HIF-1 stimulates glycolysis by activating the expression of the glucose transporters GLUT and glycolytic enzymes such as HK2, lactate dehydrogenase A, and PDK1, an inhibitor of PDH, that inhibits mitochondrial uptake of pyruvate. Mutation of p53 blocks the expression of SCO2 that is critical for regulating the COX complex, the major site of mitochondrial oxygen utilization. Despite the huge production of lactate⁻/H⁺ (represented in orange), tumour cells maintain the pHi compatible with cell viability by activating their constitutively expressed pHi-regulating system (black symbols) such as the Na⁺/H⁺ exchanger-1 (NHE-1), the bicarbonate transporters such as Cl⁻/HCO₃⁻ exchangers (AE), Na⁺-dependent HCO₃⁻ transporter (NBC), the MCT1, the H⁺-pump (V-ATPase), the cytoplasmic carbonic anhydrase II (CAII) and the HIF-1-induced pHi-regulating systems (blue symbols) such as the MCT4, carbonic anhydrases IX and XII (CAIX and CAXII) and aquaporin 1 (AQP1). Many of these components, which regulate the pHi, may interact to form a ‘metabolon’ that enhances metabolic flux of H⁺, lactate⁻ and HCO₃⁻ (represented by double arrows).

Fig 8

Cross talk between CAIX and CAXII expression. Reduced expression of CAIX by doxycycline (DOX)-induced shRNA interference in LS174Tr human colon carcinoma cells (LS-shca9) leads to up-regulation of CAXII. Co-staining for CAIX (Immunofluorescence) (upper panel) and CAXII (Immunohistochemistry) (lower panel) was observed in mice xenograft tumours of inducible colon carcinoma LS-shca9 cells with (+DOX) or without (–DOX) doxycycline in the drinking water of mice. Magnification 20×.