AICAR Antiproliferative Properties Involve the AMPK-Independent Activation of the Tumor Suppressors LATS 1 and 2.

AICAR (Acadesine) is a pharmacological precursor of purine nucleotide biosynthesis with anti-tumoral properties. Although recognized as an AMP mimetic activator of the protein kinase AMPK, the AICAR monophosphate derivative ZMP was also shown to mediate AMPK-independent effects. In order to unveil these AMPK-independent functions, we performed a transcriptomic analysis in AMPKα1/α2 double knockout murine embryonic cells. Kinetic analysis of the cellular response to AICAR revealed the up-regulation of the large tumor suppressor kinases (Lats) 1 and 2 transcripts, followed by the repression of numerous genes downstream of the transcriptional regulators Yap1 and Taz. This transcriptional signature, together with the observation of increased levels in phosphorylation of Lats1 and Yap1 proteins, suggested that the Hippo signaling pathway was activated by AICAR. This effect was observed in both fibroblasts and epithelial cells. Knockdown of Lats1/2 prevented the cytoplasmic delocalization of Yap1/Taz proteins in response to AICAR and conferred a higher resistance to the drug. These results indicate that activation of the most downstream steps of the Hippo cascade participates to the antiproliferative effects of AICAR.

Glioblastoma invasion and cooption depend on IRE1α endoribonuclease activity.

IRE1α is an endoplasmic reticulum (ER)-resident transmembrane signaling protein and a cellular stress sensor. The protein harbors a cytosolic dual kinase/endoribonuclease activity required for adaptive responses to micro-environmental changes. In an orthotopic xenograft model of human glioma, invalidation of IRE1α RNase or/and kinase activities generated tumors with remarkably distinct phenotypes. Contrasting with the extensive angiogenesis observed in tumors derived from control cells, the double kinase/RNase invalidation reprogrammed mesenchymal differentiation of cancer cells and produced avascular and infiltrative glioblastomas with blood vessel co-option. In comparison, selective invalidation of IRE1α RNase did not compromise tumor angiogenesis but still elicited invasive features and vessel co-option. In vitro, IRE1α RNase deficient cells were also endowed with a higher ability to migrate. Constitutive activation of both enzymes led to wild-type-like lesions. The presence of IRE1α, but not its RNase activity, is therefore required for glioblastoma neovascularization, whereas invasion results only from RNase inhibition. In this model, two key mechanisms of tumor progression and cancer cell survival are functionally linked to IRE1α.

Identification of yeast and human 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAr) transporters.

5-Aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAr) is the precursor of the active monophosphate form (AICAR), a small molecule with potent anti-proliferative and low energy mimetic properties. The molecular bases for AICAR toxicity at the cellular level are poorly understood. Here, we report the isolation and characterization of several yeast AICAr-hypersensitive mutants. Identification of the cognate genes allowed us to establish that thiamine transporters Thi7 and Thi72 can efficiently take up AICAr under conditions where they are overexpressed. We establish that, under standard growth conditions, Nrt1, the nicotinamide riboside carrier, is the major AICAr transporter in yeast. A study of AICAR accumulation in human cells revealed substantial disparities among cell lines and confirmed that AICAr enters cells via purine nucleoside transporters. Together, our results point to significant differences between yeast and human cells for both AICAr uptake and AICAR accumulation.

High epiregulin expression in human U87 glioma cells relies on IRE1α and promotes autocrine growth through EGF receptor.

BACKGROUND: Epidermal growth factor (EGF) receptors contribute to the development of malignant glioma. Here we considered the possible implication of the EGFR ligand epiregulin (EREG) in glioma development in relation to the activity of the unfolded protein response (UPR) sensor IRE1α. We also examined EREG status in several glioblastoma cell lines and in malignant glioma. METHODS: Expression and biological properties of EREG were analyzed in human glioma cells in vitro and in human tumor xenografts with regard to the presence of ErbB proteins and to the blockade of IRE1α. Inactivation of IRE1α was achieved by using either the dominant-negative strategy or siRNA-mediated knockdown. RESULTS: EREG was secreted in high amounts by U87 cells, which also expressed its cognate EGF receptor (ErbB1). A stimulatory autocrine loop mediated by EREG was evidenced by the decrease in cell proliferation using specific blocking antibodies directed against either ErbB1 (cetuximab) or EREG itself. In comparison, anti-ErbB2 antibodies (trastuzumab) had no significant effect. Inhibition of IRE1α dramatically reduced EREG expression both in cell culture and in human xenograft tumor models. The high-expression rate of EREG in U87 cells was therefore linked to IRE1α, although being modestly affected by chemical inducers of the endoplasmic reticulum stress. In addition, IRE1-mediated production of EREG did not depend on IRE1 RNase domain, as neither the selective dominant-negative invalidation of the RNase activity (IRE1 kinase active) nor the siRNA-mediated knockdown of XBP1 had significant effect on EREG expression. Finally, chemical inhibition of c-Jun N-terminal kinases (JNK) using the SP600125 compound reduced the ability of cells to express EREG, demonstrating a link between the growth factor production and JNK activation under the dependence of IRE1α. CONCLUSION: EREG may contribute to glioma progression under the control of IRE1α, as exemplified here by the autocrine proliferation loop mediated in U87 cells by the growth factor through ErbB1.

FTIR spectro-imaging of collagen scaffold formation during glioma tumor development.

Evidence has recently emerged that solid and diffuse tumors produce a specific extracellular matrix (ECM) for division and diffusion, also developing a specific interface with microvasculature. This ECM is mainly composed of collagens and their scaffolding appears to drive tumor growth. Although collagens are not easily analyzable by UV-fluorescence means, FTIR imaging has appeared as a valuable tool to characterize collagen contents in tissues, specially the brain, where ECM is normally devoid of collagen proteins. Here, we used FTIR imaging to characterize collagen content changes in growing glioma tumors. We could determine that C6-derived solid tumors presented high content of triple helix after 8-11 days of growth (typical of collagen fibrils formation; 8/8 tumor samples; 91 % of total variance), and further turned to larger α-helix (days 12-15; 9/10 of tumors; 94 % of variance) and ß-turns (day 18-21; 7/8 tumors; 97 % of variance) contents, which suggest the incorporation of non-fibrillar collagen types in ECM, a sign of more and more organized collagen scaffold along tumor progression. The growth of tumors was also associated to the level of collagen produced (P < 0.05). This study thus confirms that collagen scaffolding is a major event accompanying the angiogenic shift and faster tumor growth in solid glioma phenotypes.

Use of synchrotron-radiation-based FTIR imaging for characterizing changes in cell contents.

FTIR imaging of individual cells is still limited by the low signal-to-noise ratio obtained from analysis of such weakly absorbing organic matter when using a Globar IR source. In this study, we used FTIR imaging with a synchrotron radiation source and a focal plane array detector to determine changes in the cellular contents of cryofixed cells after culture for 48 h on Si(3)N(4) substrate. Several spectral differences were observed for cells deprived of glucose compared with control cells: a lower amide I-to-amide II ratio (P < 0.01); a different secondary structure profile of proteins (obtained from amide I spectral region curve fitting), with a significant increase in non-ordered structure components (P < 0.01); and a higher ν(C = C-H)/ν(as)(CH(3)) absorption ratio (P < 0.01), suggesting increased unsaturation of fatty acyl chains. Therefore, our study has shown that FTIR imaging with a synchrotron radiation source enables determination of several spectral changes of individual cells between two experimental conditions, which thus opens the way to cell biology studies with this vibrational spectroscopy technique.

FTIR spectro-imaging of collagens for characterization and grading of gliomas.

Collagens are a family of at least 30 protein types organized as networks. They constitute the main support material of cells under the form of extracellular matrix as well as for membranes in vessels, organs, and tissue compartments. Collagen network abnormalities are at the origin of many diseases, including myopathies and fibroses. The characterization of collagens remains an analytical challenge due to the insolubility of these molecules and the difficulty encountered in isolating given types without altering their structure or in maintaining network organization, which is critical to diagnosing related pathologies. We have proposed using a vibrational spectroscopy based imaging technique, namely Fourier-transform infrared (FTIR) imaging, for a spatially-resolved analysis of secondary structure of different collagen types in complex samples, and more specifically for characterizing gliomas. With newly developed spectral data treatments and chemometrics using secondary structure parameters of collagen proteins, FTIR imaging is now able to distinguish between several types. On this basis, gliomas have been investigated as specific collagen-rich tissues developing in a non-collagenous environment, providing high specificity to this FTIR imaging utilization. Here, we review the recent advances in this imaging approach for understanding glioma development, with FTIR imaging now being proposed as a molecular histopathology tool for clinicians.

Inhibition of angiogenesis and the angiogenesis/invasion shift.

Angiogenesis has become a major target in cancer therapy. However, current therapeutic strategies have their limitations and raise several problems. In most tumours, anti-angiogenesis treatment targeting VEGF (vascular endothelial growth factor) has only limited overall survival benefit compared with conventional chemotherapy alone, and reveals several specific forms of resistance to anti-VEGF treatment. There is growing evidence that anti-VEGF treatment may induce tumour cell invasion by selecting highly invasive tumour cells or hypoxia-resistant cells, or by up-regulating angiogenic alternative pathways such as FGFs (fibroblast growth factors) or genes triggering new invasive programmes. We have identified new genes up-regulated during glioma growth on the chick CAM (chorioallantoic membrane). Our results indicate that anti-angiogenesis treatment in the experimental glioma model drives expression of critical genes which relate to disease aggressiveness in glioblastoma patients. We have identified a molecular mechanism in tumour cells that allows the switch from an angiogenic to invasive programme. Furthermore, we are focusing our research on alternative inhibitors that act, in part, independently of VEGF. These are endogenous molecules that play a role in the control of tumour growth and may constitute a starting point for further development of novel therapeutic or diagnostic tools.

Peptides derived from the bifunctional kinase/RNase enzyme IRE1α modulate IRE1α activity and protect cells from endoplasmic reticulum stress.

Activation of the bifunctional kinase/RNase enzyme IRE1α is part of an adaptive response triggered on accumulation of misfolded proteins in the endoplasmic reticulum (ER). To facilitate recovery of ER homeostasis, IRE1α molecules oligomerize, allowing for their transautophosphorylation and endoribonuclease activation. These, in turn, induce the activation of specific transcriptional and post-transcriptional programs. To identify novel and selective modulators of IRE1α activity, we investigated IRE1α oligomerization properties using IRE1α-derived peptides identified through an activity-based in vitro assay. We then used these peptides to probe IRE1α activity in vitro and in vivo using both cultured human hepatocellular carcinoma-derived HuH7 cells and Caenorhabditis elegans experimental systems. We identified a peptide derived from the kinase domain of human IRE1α, which promoted IRE1α oligomerization in vitro, enhanced its Xbp1 mRNA cleavage activity in vitro (1.7×) in cell culture (1.8×) and in vivo (1.3×), and attenuated both ER stress-mediated JNK activation and regulated IRE1-dependent mRNA decay (RIDD). This was accompanied by a 2.5-fold increase in survival on tunicamycin-induced ER stress and reduced apoptosis by 1.4-fold in cells expressing this peptide. Hence, targeted and selective activation of the catalytic properties of IRE1α may consequently define new strategies to protect cells from deleterious effects of ER stress signaling.

Functional histology of glioma vasculature by FTIR imaging.

Fourier-transform infrared (FTIR) imaging has been used to investigate brain tumor angiogenesis using a mice solid tumor model and bare-gold (∅ 25 nm) or BaSO(4) (∅ 500 nm) nanoparticles (NP) injected into blood vasculature. FTIR images of 20-µm-thick tissue sections were used for chemical histology of healthy and tumor areas. Distribution of BaSO(4)-NP (using the 1,218-1,159 cm(-1) spectral interval) revealed clearly all details of blood vasculature with morphological abnormalities of tumor capillaries, while Au-NP (using the 1,046-1,002 cm(-1) spectral interval) revealed also diffusion properties of leaky blood vessels. Diffusion of Au-NP out of vascular space reached 64 ± 29 µm, showing the fenestration of "leaky" tumor blood vessels, which should allow small NP (<100 nm, as for Au-NP) to diffuse almost freely, while large NP should not (as for BaSO(4)-NP in this study). Therefore, we propose to develop FTIR imaging as a convenient tool for functional molecular histology imaging of brain tumor vasculature, both for identifying blood capillaries and for determining the extravascular diffusion space offered by vessel fenestration.

Detection of collagens in brain tumors based on FTIR imaging and chemometrics.

Fourier transform infrared (FTIR) imaging has been used as a molecular histopathology tool on brain tissue sections after intracranial implantation and development of glioma tumors. Healthy brain tissue (contralateral lobe) as well as solid and diffuse tumor tissues were compared for their collagen contents. IR spectra were extracted from IR images for determining the secondary structure of protein contents and compared to pure product spectra of collagens (types I, III, IV, V, and VI). Multivariate statistical analyses of variance and correspondence factorial analysis were performed to differentiate healthy and tumor brain tissues as well as their classification according to their secondary structure profiles. Secondary structure profiles revealed that no collagen was present in healthy tissues; they are also significantly different from solid and diffuse tumors (p < 0.05). Solid and diffuse tumors could be discriminated with respect to the secondary structure profile of fibrillar and non-fibrillar collagens, respectively. We can thus propose to develop FTIR imaging for histopathology examination of tumors on the basis of collagen contents.

"Click" conjugation of peptide on the surface of polymeric nanoparticles for targeting tumor angiogenesis.

PURPOSE: Angiogenesis plays a critical role in tumor growth. This phenomena is regulated by numerous mediators such as vascular endothelial growth factor (VEGF). CBO-P11, a cyclo-peptide, has proven to specifically bind to receptors of VEGF and may be used as targeting ligand for tumor angiogenesis. We herein report the design of novel nanoparticles conjugated to CBO-P11 in order to specifically target tumor site. METHODS: The conjugation of CBO-P11 on the surface of poly(vinylidene fluoride) (PVDF) nanoparticles was investigated using the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition known as "click" reaction. CBO-P11 was modified with a near-infrared cyanine dye bearing an alkyne function, allowing both "click" coupling on azido-modified nanoparticles and fluorescence labelling. Each step of this nanodevice construction was judiciously performed in aqueous solution and successfully characterized. The cytotoxicity of nanoparticles was evaluated in human brain endothelial cell line and their affinity for VEGF receptors was determined via fluorescence-based uptake assays on porcine aortic endothelial cell line. RESULTS: Nanoparticles were found to be spherical, dense, monodisperse and stable. No cytotoxicity was observed after four days of incubation demonstrating the biocompatibility of nanoparticles. Fluorescence highlighted the specific interaction of these functionalized nanoparticles for VEGF receptors, suggesting that the targeting peptide bioactivity was retained. CONCLUSIONS: These results demonstrate the potential of these functionalized nanoparticles for targeting tumor angiogenesis and their possible use as multifunctional platform for cancer treatment if coupled with therapeutic agents.

Inositol-requiring enzyme 1alpha is a key regulator of angiogenesis and invasion in malignant glioma.

Inositol-requiring enzyme 1 (IRE1) is a proximal endoplasmic reticulum (ER) stress sensor and a central mediator of the unfolded protein response. In a human glioma model, inhibition of IRE1alpha correlated with down-regulation of prevalent proangiogenic factors such as VEGF-A, IL-1beta, IL-6, and IL-8. Significant up-regulation of antiangiogenic gene transcripts was also apparent. These transcripts encode SPARC, decorin, thrombospondin-1, and other matrix proteins functionally linked to mesenchymal differentiation and glioma invasiveness. In vivo, using both the chick chorio-allantoic membrane assay and a mouse orthotopic brain model, we observed in tumors underexpressing IRE1: (i) reduction of angiogenesis and blood perfusion, (ii) a decreased growth rate, and (iii) extensive invasiveness and blood vessel cooption. This phenotypic change was consistently associated with increased overall survival in glioma-implanted recipient mice. Ectopic expression of IL-6 in IRE1-deficient tumors restored angiogenesis and neutralized vessel cooption but did not reverse the mesenchymal/infiltrative cell phenotype. The ischemia-responsive IRE1 protein is thus identified as a key regulator of tumor neovascularization and invasiveness.

FT-IR spectral imaging of blood vessels reveals protein secondary structure deviations induced by tumor growth.

Vascular basement membrane remodeling is involved in tumor angiogenesis to enable tumor invasion and growth. FT-IR spectral imaging was used to determine changes in tumor blood vessels to reveal protein secondary structure in Rag-gamma immuno-deficient mice sacrificed 14 and 21 days after subcutaneous glioma implantation. For the oldest blood capillaries (diameter >20 microns), tumor growth induced a decrease in triple-helix content (1638 cm(-1); -7.3%; P < 0.05) and an increase in beta turns (1666 and 1615 cm(-1); +4%; P < 0.01). These protein-structure alterations, mainly from type IV collagen, reflected the high angiogenic stress of growing tumors. We propose to use these molecular markers of vascular basement membrane protein alterations for gradation of solid tumors by FT-IR spectral imaging.

Integrated endoplasmic reticulum stress responses in cancer.

The endoplasmic reticulum (ER) has emerged as a major site of cellular homeostasis regulation, particularly in the unfolded protein response, which is being found to play a major role in cancer and many other diseases. Here, we address ER-mediated signaling and regulations in the context of environmental challenges in cancer, such as hypoxia, angiogenesis, and chemotherapeutic resistance, and we discuss how ER-resident molecular machines become deregulated and involved in cancer-related pathology. Further exploration of how the ER senses, signals, and adapts to stress may redefine and deepen our understanding of its functions in cancer pathobiology.

Histological mapping of biochemical changes in solid tumors by FT-IR spectral imaging.

Fourier-transform infrared (FT-IR) spectral imaging was used for analyzing biochemical changes in tumor cells. Metabolic parameters of human lung A549/8 adenocarcinoma and U87 glioma cells were compared under stress conditions in culture along with tumor progression after cell implantation onto the chick embryo chorio-allantoic membrane. In cell culture, glucose consumption and lactic acid release were higher in U87 cells. A549/8 cells were less sensitive to oxidative stress as observed through changes in fatty acyl chains. In vivo biochemical mapping of highly (U87) vs. poorly (A549/8) angiogenic tumors provided results comparable to culture models. Therefore, FT-IR imaging allows detecting subtle chemical changes in tumors, which might be useful for diagnosis.

IRE1 signaling is essential for ischemia-induced vascular endothelial growth factor-A expression and contributes to angiogenesis and tumor growth in vivo.

In solid tumors, cancer cells subjected to ischemic conditions trigger distinct signaling pathways contributing to angiogenic stimulation and tumor development. Characteristic features of tumor ischemia include hypoxia and glucose deprivation, leading to the activation of hypoxia-inducible factor-1-dependent signaling pathways and to complex signaling events known as the unfolded protein response. Here, we show that the activation of the endoplasmic reticulum stress sensor IRE1 is a common determinant linking hypoxia- and hypoglycemia-dependent responses to the up-regulation of vascular endothelial growth factor-A (VEGF-A). Tumor cells expressing a dominant-negative IRE1 transgene as well as Ire1alpha-null mouse embryonic fibroblasts were unable to trigger VEGF-A up-regulation upon either oxygen or glucose deprivation. These data correlated with a reduction of tumor angiogenesis and growth in vivo. Our results therefore suggest an essential role for IRE1-dependent signaling pathways in response to ischemia and identify this protein as a potential therapeutic target to control both the angiogenic switch and tumor development.

Acute L-glutamine deprivation compromises VEGF-a upregulation in A549/8 human carcinoma cells.

Tumor ischemia participates in angiogenesis and cancer progression through cellular responses to hypoxia and nutrient deprivation. However, the contribution of amino acids limitation to this process remains poorly understood. Using serum-free cell culture conditions, we tested the impact of L-glutamine deprivation on metabolic and angiogenic responses in A549/8 carcinoma cells. In these cells, lowering glutamine concentration modified the cell cycle distribution and significantly induced apoptosis/necrosis. Although glutamine deprivation led to a HIF-independent increase in VEGF-A mRNA, the corresponding protein level remained low and correlated with the inhibition of protein synthesis and activation of the GCN2/eIF2alpha pathway. Limitation of glutamine availability also hampers hypoxia- and hypoglycemia-induced VEGF-A protein upregulation. Thus, glutamine deprivation may have no direct effect on VEGF-dependent angiogenesis, compared to hypoxia or to glucose deprivation, and may instead be detrimental to cancer progression by antagonizing ischemia-induced stresses.

Glycosylation of serum ribonuclease 1 indicates a major endothelial origin and reveals an increase in core fucosylation in pancreatic cancer.

Human pancreatic ribonuclease 1 (RNase 1) is a glycoprotein expressed mainly by the pancreas and also found in endothelial cells. The diagnosis of pancreatic cancer (PaC) remains difficult and therefore the search for sensitive and specific markers is required. Previous studies showed that RNase 1 from human healthy pancreas contained only neutral glycans, whereas RNase 1 from PaC cell lines contained sialylated structures. To determine whether these glycan tumor cell-associated changes were also characteristic of serum RNase 1 and could be used as a marker of PaC, we have analyzed the glycosylation of serum RNase 1. The origin of serum RNase 1 was also investigated. Serum RNase 1 from two PaC patients and two controls was purified and the glycans analyzed by high-performance liquid chromatography (HPLC)-based sequencing and mass spectrometry. Although normal and tumor serum RNase 1 contained the same glycan structures, there was an increase of 40% in core fucosylation in the main sialylated biantennary glycans in the PaC serum RNase 1. This change in proportion would be indicative of a subset of tumor-associated glycoforms of RNase 1, which may provide a biomarker for PaC. Two-dimensional electrophoresis of the RNase 1 from several endothelial cell lines, EA.hy926, human umbilical vein endothelial cells (HUVEC), human mammary microvessel endothelial cells (HuMMEC), and human lung microvessel endothelial cells (HuLEC), showed basically the same pattern and was also very similar to that of serum RNase 1. RNase 1 from EA.hy926 was then purified and presented a glycosylation profile very similar to that from serum RNase 1, suggesting that endothelial cells are the main source of this enzyme.

Hypoxic regulation of PFKFB-3 and PFKFB-4 gene expression in gastric and pancreatic cancer cell lines and expression of PFKFB genes in gastric cancers.

Previously we have shown that hypoxia strongly induces the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and -4 (PFKFB-3 and PFKFB-4) genes in several cancer cell lines via a HIF-dependent mechanism. In this paper we studied the expression and hypoxic regulation of PFKFB-4 and PFKFB-3 mRNA as well as its correlation with HIF-1alpha, HIF-2alpha, VEGF and Glut1 mRNA expression in the pancreatic cancer cell line Panc1 and two gastric cancer cell lines MKN45 and NUGC3. This study clearly demonstrated that PFKFB-3 and PFKFB-4 mRNA are expresses in MKN45, NUGC3 and Panc1 cancers cells and that both genes are responsive to hypoxia in vitro. However, their basal level of expression and hypoxia responsiveness vary in the different cells studied. Particularly, PFKFB-3 mRNA is highly expressed in MKN45 and NUGC3 cancer cells, with the highest response to hypoxia in the NUGC3 cell line. The PFKFB-4 mRNA has a variable low basal level of expression in both gastric and pancreatic cancer cell lines. However, the highest hypoxia response of PFKFB-4 mRNA is found in the pancreatic cancer cell line Panc1. The basal level of PFKFB-4 protein expression is the highest in NUGC3 gastric cancer cell line and lowest in Panc1 cells, with the highest response to hypoxia in the pancreatic cancer cell line. Further studies showed that PFKFB-3 and PFKFB-4 gene expression was highly responsive to the hypoxia mimic dimethyloxalylglycine, a specific inhibitor of HIF-alpha hydroxylase enzymes, suggesting that the hypoxia responsiveness of PFKFB-3 and PFKFB-4 genes in these cell lines is regulated by the HIF transcription complex. The expression of VEGF and Glut1, which are known HIF-dependent genes, is also strongly induced under hypoxic conditions in gastric and pancreatic cancer cell lines. The levels of HIF-1alpha protein are increased in both gastric and pancreatic cancer cell lines under hypoxic conditions. However, the basal level of HIF-1alpha as well as HIF-2alpha mRNA expression and their hypoxia responsiveness are different in the MKN45 and NUGC3 cancer cells. Thus, the expression of HIF-1alpha mRNA is decreased in both gastric cancer cell lines treated by hypoxia or dimethyloxalylglycine, but HIF-2alpha mRNA expression is not changed significantly in NUGC3 and slightly increased in MKN45 cells. Expression of PFKFB-4 and PFKFB-3 was also studied in gastric cancers and corresponding nonmalignant tissue counterparts from the same patients on both the mRNA and protein levels. The expression of PFKFB-3 and PFKFB-4 mRNA as well as PFKFB-1 and PFKFB-2 mRNA was observed in normal human gastric tissue and was increased in malignant gastric tumors. The basal level of PFKFB-4 protein expression in gastric cancers was much higher as compared to the PFKFB-3 isoenzyme. In conclusion, this study provides evidence that PFKFB-4 and PFKFB-3 genes are also expressed in gastric and pancreatic cancer cells, they strongly respond to hypoxia via a HIF-1alpha dependent mechanism and, together with the expression of PFKFB-1 and PFKFB-2 genes, possibly have a significant role in the Warburg effect which is found in malignant cells.