Adaptation to HIF-1 deficiency by upregulation of the AMP/ATP ratio and phosphofructokinase activation in hepatomas.

BACKGROUND: HIF-1 deficiency has marked effects on tumour glycolysis and growth. We therefore investigated the consequences of HIF-1 deficiency in mice, using the well established Hepa-1 wild-type (WT) and HIF-1ß-deficient (c4) model. These mechanisms could be clinically relevant, since HIF-1 is now a therapeutic target. METHODS: Hepa-1 WT and c4 tumours grown in vivo were analysed by 18FDG-PET and 19FDG Magnetic Resonance Spectroscopy for glucose uptake; by HPLC for adenine nucleotides; by immunohistochemistry for GLUTs; by immunoblotting and by DIGE followed by tandem mass spectrometry for protein expression; and by classical enzymatic methods for enzyme activity. RESULTS: HIF-1ß deficient Hepa-1 c4 tumours grew significantly more slowly than WT tumours, and (as expected) showed significantly lower expression of many glycolytic enzymes. However, HIF-1ß deficiency caused no significant change in the rate of glucose uptake in c4 tumours compared to WT when assessed in vivo by measuring fluoro-deoxyglucose (FDG) uptake. Immunohistochemistry demonstrated less GLUT-1 in c4 tumours, whereas GLUT-2 (liver type) was similar to WT. Factors that might upregulate glucose uptake independently of HIF-1 (phospho-Akt, c-Myc) were shown to have either lower or similar expression in c4 compared to WT tumours. However the AMP/ATP ratio was 4.5 fold higher (p < 0.01) in c4 tumours, and phosphofructokinase-1 (PFK-1) activity, measured at prevailing cellular ATP and AMP concentrations, was up to two-fold higher in homogenates of the deficient c4 cells and tumours compared to WT (p < 0.001), suggesting that allosteric PFK activation could explain their normal level of glycolysis. Phospho AMP-Kinase was also higher in the c4 tumours. CONCLUSIONS: Despite their defective HIF-1 and consequent down-regulation of glycolytic enzyme expression, Hepa-1 c4 tumours maintain glucose uptake and glycolysis because the resulting low [ATP] high [AMP] allosterically activate PFK-1. This mechanism of resistance would keep glycolysis functioning and also result in activation of AMP-Kinase and growth inhibition; it may have major implications for the therapeutic activity of HIF inhibitors in vivo. Interestingly, this control mechanism does not involve transcriptional control or proteomics, but rather the classical activation and inhibition mechanisms of glycolytic enzymes.

Glut-1 as a therapeutic target: increased chemoresistance and HIF-1-independent link with cell turnover is revealed through COMPARE analysis and metabolomic studies.

The facilitative glucose transporter Glut-1 is overexpressed and confers poor prognosis in a wide range of solid tumours. The peri-necrotic pattern of expression often seen in human tumour samples is linked with its transcriptional control in hypoxic conditions by hypoxia-inducible factor HIF-1 or through a reduced rate of oxidative phosphorylation. Hypoxia-regulated genes offer promise as novel therapeutic targets as a means of preventing the proliferation and eventual metastatic spread of tissue originating from residual chemically and radio resistant hypoxic cells that have survived treatment. Inhibiting the expression or functionality of Glut-1 may be a way of specifically targeting hypoxic cells within the tumour that depend upon a high rate of glucose uptake for anaerobic glycolysis. We used an array of formalin-fixed, paraffin-embedded samples of the NCI-60 panel of cell lines to carry out immunohistochemical detection of Glut-1 and to select possible candidate lead compounds by COMPARE analysis with agents from the NCI diversity screen, which may work via inhibition of Glut-1 or Glut-1-dependent processes. "Positive" COMPARE hits were mostly conjugated Pseudomonas toxins binding the epidermal growth factor receptor (EGFR). However, correlations with standard anticancer agents were virtually all negative, indicating a link between Glut-1 and chemoresistance. MTT proliferation assays carried out using stable, Glut-1 overexpressing cell lines generated from the bladder EJ138, human fibrosarcoma HT 1080 and the hepatoma wild type Hepa and HIF-1B-deficient c4 tumour cell lines revealed a cell line-dependent increase in chemoresistance to dacarbazine, vincristine and the bioreductive agent EO9 in Glut-1 overexpressing EJ138 relative to WT and empty vector controls. Metabolomic analysis ((31)P-MRS and (1)H MRS) carried out using cell lysates and xenografts generated from Glut-1 overexpressing Hepa and c4 cell lines showed higher glucose levels in Glut-1 overxpressing c4 relative to parental tumour extracts occurred in the absence of an increase in lactate levels, which were in turn significantly higher in the Glut-1 overexpressing Hepa xenografts. This implies that Glut-1 over-expression without a co-ordinate increase in HIF-1-regulated glycolytic enzymes increases glucose uptake but not the rate of glycolysis. Glut-1 overexpressing xenografts also showed higher levels of phosphodiester (PDE), which relates to the metabolite turnover of phospholipids and is involved in membrane lipid degradation, indicating a mechanism by which Glut-1 may increase cell turnover.

Hypoxic regulation of glucose transport, anaerobic metabolism and angiogenesis in cancer: novel pathways and targets for anticancer therapeutics.

Cancer cells require a steady source of metabolic energy in order to continue their uncontrolled growth and proliferation. Accelerated glycolysis is one of the biochemical characteristics of cancer cells. Recent work indicates that glucose transport and metabolism are essential for the posttreatment survival of tumor cells, leading to poor prognosis. Glycolytic breakdown of glucose is preceded by the transport of glucose across the cell membrane, a rate-limiting process mediated by facilitative glucose transporter proteins belonging to the facilitative glucose transporter/solute carrier GLUT/SLC2A family. Tumors frequently show overexpression of GLUTs, especially the hypoxia-responsive GLUT1 and GLUT3 proteins. There are also studies that have reported associations between GLUT expression and proliferative indices, whilst others suggest that GLUT expression may be of prognostic significance. In this article we revisit Warburg's original hypothesis and review the recent clinical and basic research on the expression of GLUT family members in human cancers and in cell lines derived from human tumors. We also explore the links between hypoxia-induced genes, glucose transporters and angiogenic factors. Hypoxic tumors are significantly more malignant, metastatic, radio- and chemoresistant and have a poor prognosis. With the discovery the oxygen-sensitive transcription factor hypoxia-inducible factor (HIF-1) has come a new understanding of the molecular link between hypoxia and deregulated glucose metabolism. HIF-1 induces a number of genes integral to angiogenesis, e.g. vascular endothelial growth factor (VEGF), a process intimately involved with metastatic spread. This knowledge may enhance existing chemotherapeutic strategies so that treatment can be more rationally applied and personalized for cancer patients.

A protective role for HIF-1 in response to redox manipulation and glucose deprivation: implications for tumorigenesis.

We have investigated the role of HIF-1 in the cellular response to redox modulation via the inhibition of oxidative phosphorylation. We demonstrate that manipulation of redox in air, achieved by inhibiting cytochrome oxidase with cyanide, induces HIF-1 mediated transcription in wild-type CHO and HT1080 human tumour cells but not in CHO cells deficient in the oxygen responsive, HIF-1alpha sub-unit of HIF-1. Hypoglycaemia attenuates cyanide-mediated transcription in non-transformed HIF-1 wild-type CHO cells but not the human tumour derived cell line. Cells lacking either HIF-1alpha, or the second composite sub-unit of HIF-1, HIF-1beta, were markedly more sensitive to the combined stress of perturbed redox and hypoglycaemia than wild-type cells. As such conditions together with hypoxia are prevalent in tumours, these data suggest that HIF-1 may have a protective role in adaptation to the tumour micro-environment. In support of this we demonstrate that HIF-1alpha deficient cells are less tumorigenic than wild-type cells. They showed a reduced growth rate when grown as xenografts in nude mice. This was not related to vascular parameters that were identical to those found in HIF-1 wild-type tumours. The HIF-1 deficient tumours lacked focal expression of Glut-1 in hypoxic tumour regions. Compromised glucose uptake and metabolic adaptation to the tumour micro-environment may form the basis of the reduced tumorigenicity associated with these cells.

Heterogeneous expression of the aquaporin 1 (AQP1) water channel in tumors of the prostate, breast, ovary, colon and lung: a study using high density multiple human tumor tissue microarrays.

Aquaporin 1 (AQP1) water channels are membrane proteins that control the permeability of endothelial and epithelial barriers by facilitating water movement across cell membranes. Recent studies suggest that AQP1 may be responsible for the high vascular permeability and interstitial fluid pressure in tumors of the brain, colon, breast and pancreas. AQP1 may also play a role in tumor angiogenesis and may be involved in development of effusions or edema fluid. The aim of the present study was to use immunohistochemistry and semi-quantitative histomorphometric analysis to compare the distribution and relative abundance of AQP1 on NCI TARP human multiple tumor tissue microarrays (TMAs) with normal tissues represented on the CHTN TMAs. Immunohistochemistry and semi-quantitative histomorphometric analysis were used to compare the distribution of AQP1 in tumors of the prostate, colon, lung, breast and ovary represented on TARP TMAs with their normal counterparts on CHTN TMAs. AQP1 was expressed in capillary endothelia of all normal tissues. In most tumors AQP1 was confined to endothelial barriers. AQP1 expression was marginally higher in microvascular structures in prostate and ovarian tumors and was higher in advanced mammary and colorectal carcinomas where AQP1 immunoreactivity was also seen in some neoplastic tumor cells. In conclusion, the AQP1 water channel is an excellent marker of microvasculature but it is heterogeneously expressed in different human tumors and not necessarily expressed in all neoplastic cells. Increased AQP1 expression in some human adenocarcinomas may be a consequence of angiogenesis and important for the formation or clearance of tumor edema.

Viral delivery of P450 reductase recapitulates the ability of constitutive overexpression of reductase enzymes to potentiate the activity of mitomycin C in human breast cancer xenografts.

Indolequinones such as mitomycin C (MMC) require enzymatic bioreduction to yield cytotoxic moieties. An attractive approach to overcome the potential variability in reductive bioactivation between tumors is to exploit specific enzyme-bioreductive drug combinations in an enzyme-directed gene therapy (GDEPT) approach. To this end, human breast cancer cell lines (T47D, MDA468, and MDA231) that overexpress either DT-diaphorase (DTD) or NADPH:cytochrome P450 reductase (P450R) have been developed. Cytotoxicity of MMC was evaluated in the panel of cell lines following aerobic or anoxic exposure in vitro. DTD and/or P450R overexpression sensitized cells to MMC in air with no further increase in the cytotoxicity of MMC under anoxia. The most profound effect was seen in the MDA468 cells, where a 27-fold increase in potency was observed for MMC in the DTD-overexpressing cell line. The MMC sensitization achieved through DTD and P450R overexpression in MDA468 cells was maintained in vivo. Xenografts established from the clonal lines exhibited significant tumor control following MMC treatment (treated/control [T/C] 17% and 51% for DTD and P450R xenografts, respectively) that was not seen in wild-type tumors (T/C 102%). Delivery of a clinically relevant adenoviral vector encoding P450R to MDA468 wild-type tumors yielded comparable P450R activity to that seen in the P450R clonal xenografts and resulted in greater MMC sensitization (T/C 46%). The model systems developed will facilitate the identification of novel indolequinone agents that are targeted toward a specific enzyme for bioactivation and are consequently of potential use in a GDEPT approach.

Could GLUT12 be a Potential Therapeutic Target in Cancer Treatment? A Preliminary Report.

BACKGROUND: Recent studies proposed GLUT12 to be a major glucose transporter involved in the glycolytic metabolism of cancer cells. METHODS: GLUT12 expression was determined by immunohistochemistry in a selection of cancer cell lines and a tumour spheroid model. RESULTS: GLUT12 expression was high in A549 and RH-36; low in HT29; and absent in NB-EB cancer cell lines. GLUT12 expression was located in the necrotic centre of HT29 spheroids, which is characterised by anaerobic metabolism. CONCLUSION: The data supports the involvement of GLUT12 in the glycolytic metabolism of cancer cells and therefore, its potential as a novel therapeutic target for cancer treatment.

Glucose transporter Glut-1 is detectable in peri-necrotic regions in many human tumor types but not normal tissues: Study using tissue microarrays.

The hypoxic tumor microenvironment is associated with malignant progression and poor treatment response. The glucose transporter Glut-1 is a prognostic factor and putative hypoxia marker. So far, studies of Glut-1 in cancer have utilized conventional immunohistochemical analysis in a series of individual biopsy or surgical specimens. Tissue microarrays, however, provide a rapid, inexpensive means of profiling biomarker expression. To evaluate hypoxia markers, tissue cores must show the architectural features of hypoxia; i.e. viable tissue surrounding necrotic regions. Glut-1 may be a useful biomarker to validate tissue microarrays for use in studies of hypoxia-regulated genes in cancer. In this study, we carried out immunohistochemical detection of Glut-1 protein in many tumor and normal tissue types in a range of tissue microarrays. Glut-1 was frequently found in peri-necrotic regions, occurring in 9/34 lymphomas, 6/12 melanomas, and 5/16 glioblastomas; and in 43/54 lung, 22/84 colon, and 23/60 ovarian tumors. Expression was rare in breast (6/40) and prostate (1/57) tumors, and in normal tissue, was restricted to spleen, tongue, and CNS endothelium. In conclusion, tissue microarrays enable the observation of Glut-1 expression in peri-necrotic regions, which may be linked to hypoxia, and reflect previous studies showing differential Glut-1 expression across tumor types and non-malignant tissue.

GLUT-1 and CAIX as intrinsic markers of hypoxia in carcinoma of the cervix: relationship to pimonidazole binding.

The presence of hypoxia in tumours results in the overexpression of certain genes, which are controlled via the transcription factor HIF-1. Hypoxic cells are known to be radioresistant and chemoresistant, thus, a reliable surrogate marker of hypoxia is desirable to ensure that treatment may be rationally applied. Recently, the HIF-1-regulated proteins Glut-1 and CAIX were validated as intrinsic markers of hypoxia by comparison with pO(2) measured using oxygen electrodes. We compare the expression of Glut-1 and CAIX with the binding of the bioreductive drug hypoxia marker pimonidazole. Pimonidazole was administered to 42 patients with advanced carcinoma of the cervix, 16 hr before biopsy. Sections of single or multiple biopsies were then immunostained for Glut-1 and CAIX, and the area of staining scored by eye, using a "field-by-field" semi-quantitative averaging system. Using 1 biopsy only, Glut-1 (r = 0.54, p = <0.001) correlated with the level of pimonidazole binding, and Glut-1 and CAIX expression also correlated significantly (r = 0.40, p = <0.009). Thus, our study has shown that HIF-1 regulated genes have potential for future use as predictors of the malignant changes mediated by hypoxia, and warrant further investigation as indicators of response to cancer therapy.