Donald W. Bowden, Center for Human Genomics, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA. e-mail: firstname.lastname@example.org
To investigate the differences in the pattern of glucose transporter (GLUT) gene expression between normal and tumour tissues and among histological subtypes of renal cell carcinomas (RCCs), as malignant cells are characterized by increased glucose uptake and use.
MATERIALS AND METHODS
Enhanced glucose uptake probably depends on the overexpression of GLUT, usually GLUT1 and/or GLUT3, but there are few comprehensive studies to evaluate the relative expression pattern and level of GLUT in normal and tumour kidney tissues, especially of the recently identified GLUT genes. In all, 71 kidney surgical samples were evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR) for GLUT1–14 in normal and tumour (clear cell, papillary and chromophobe RCC, and oncocytoma) tissues. The expression levels for GLUT1–5, 9, 10 and 12 were quantified by real-time quantitative PCR.
The RT-PCR results showed that normal kidney tissue expresses all the GLUT isoforms. In clear cell RCC GLUT1 expression increased (P < 0.001) while GLUT4, 9 and 12 decreased (P < 0.001). In papillary RCC there were no significant increases in GLUT expression, with only GLUT12 significantly expressed at lower levels (P < 0.001). In chromophobe RCC the expression of GLUT4 increased (P < 0.05), and GLUT2 and 5 decreased (P < 0.01), whereas in oncocytoma tissue there were no significant changes in the expression of GLUT1 (P < 0.01), 2, 5, 9 (P < 0.001) and 10 (P < 0.05).
These results suggest that high-affinity GLUTs might have a major role in enhanced glucose uptake in kidney tumours, and that histopathological types are characterized by specific patterns of GLUT expression.
Glucose metabolism is a central component of living systems; the oxidation of glucose represents a major source of metabolic energy for mammalian cells. However, tumour cells have a reduced ability to use oxidative metabolism and rely instead on an increased rate of glycolysis and glucose use [1,2]. A more active glycolytic metabolism is reflected in an increased rate of glucose uptake . This differential glucose uptake between normal and tumour cells is the basis for the diagnosis of various tumours by positron emission tomography using the glucose analogue 18F-fluorodeoxyglucose as a radiotracer .
As the plasma membrane is impermeable to polar molecules such as glucose, glucose uptake necessitates the use of membrane-associated carrier proteins. The facilitative glucose transporters (GLUTs) use existing gradients in glucose (and other hexoses/polyols) concentration between the external and internal faces of a membrane to facilitate their translocation, thus ensuring a continuous supply of glucose to most tissues . Initially, the GLUT family included four well-defined glucose transporters (GLUT1–4) and one fructose transporter (GLUT5) in humans. New transporters have been discovered in the past several years using informatics-based data mining of the human genome sequence; there are currently 14 GLUTs.
It is currently accepted that the increase in glucose uptake by malignant cells is associated with the overexpression of GLUTs . GLUT1 overexpression has been described in liver , pancreas , kidney , lung , breast , stomach , head and neck , and thyroid cancers . GLUT3 is overexpressed in lung, ovarian and gastric cancers . In addition to these well-defined GLUTs, a few studies recently investigated the expression of the novel GLUTs in human breast [15,16], prostate [16,17] and thyroid [13,16] tissues. However, in most cases expression patterns and levels of GLUT isoforms have not been systematically evaluated. In the present study, we examined the differences in mRNA expression patterns of all the 14 GLUT genes in normal kidney tissues and in RCCs. We also compared the relative mRNA expression levels of selected GLUT isoforms in normal and various histological subtypes of RCCs.
MATERIALS AND METHODS
Kidney samples from 71 patients who had had surgery at Wake Forest University Baptist Medical Center between 2004 and 2005 were entered into the study. Internal Review Board approval was obtained for tissue banking of all cases. Samples were snap-frozen in liquid nitrogen and stored at − 80 °C until needed. The histopathology of the tumours was classified according to the Union Internationale Contre le Cancer and the American Joint Committee on Cancer, and summarized in Table 1.
Table 1. The characteristics of the kidney samples
Number of samples (n paired samples)
clear cell RCC
For RNA extraction and cDNA synthesis, total RNA was isolated from frozen specimens with the acid-guanidinium thiocyanate-phenol-chloroform method with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Genomic DNA was removed by DNase digestion and purification through RNeasy Mini Kit columns (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The RNA quality was confirmed by agarose gel electrophoresis with ethidium bromide staining, and the 18S and 28S RNA bands were visualized under ultraviolet illumination. The yields were quantified spectrophotometrically. Single-stranded cDNA was synthesized from 1 µg of total RNA in a final volume of 20 µL containing 20 mm Tris-HCl (pH 8.4), 50 mm KCl, 5 mm MgCl2, 500 µm dNTP mix, 50 µg random hexamers and 200 units of Super Script III Reverse Transcriptase (Invitrogen) at 50 °C for 50 min. After digestion with 2 units of RNase H, 1 µg of cDNA was used as the template for PCR. mRNA samples incubated in the absence of reverse transcriptase (RT) served as negative controls.
For conventional RT-PCR the cDNA equivalent of 50 ng of total RNA was amplified with 200 µm of gene-specific primer pairs for GLUT1–14, or β-actin cDNAs (Table 2) in a 25-µL volume containing 20 mm Tris-HCl (pH 8.4), 50 mm KCl, 1.5 mm MgCl2, 200 µm dNTP mix, and 2 units of platinum Taq DNA polymerase (Invitrogen). After an initial denaturation step at 95 °C for 2 min, 40 cycles of amplification were performed at 95 °C for 30 s, 52–60 °C for 30 s and 72 °C for 30 s, followed by 3 min at 72 °C as an additional extension step in RT-PCR. For semiquantitative RT-PCR, the number of PCR cycles was adjusted for each GLUT-gene target to obtain a more accurate comparison between samples. Human adult cDNA from normal kidney, colon and testis (BioChain, Hayward, CA, USA) were used to examine the expression of GLUT7 and 14. PCR products were analysed by electrophoresis on 1.2% agarose gels and visualized by ethidium bromide staining.
Table 2. Oligonucleotide primer pairs used to detect GLUT isoforms and β-actin
GenBank accession no.
Amplicon size (bp)
All amplicons were sequenced for their position along the mRNA. aGLUT9 transcript variant 1,bGLUT9 transcript variant 2
Relative mRNA expression levels for GLUT1–5, 9, 10, and 12 genes were determined by real-time quantitative PCR. Reactions were performed with the ABI PRISM 7000 Sequence Detection System according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). All the reagents including TaqMan Primers and Probes for GLUT1–5, 9, 10, 12, endogenous β-actin control (TaqMan Assays-on-Demand Gene Expression Products; GLUT1: Hs-00197884_m1; GLUT2: Hs-00165775_m1; GLUT3: Hs-00359840_m1; GLUT4: Hs-00168966_m1; GLUT5: Hs-00161720_m1; GLUT9: Hs-00252242_m1; GLUT10: Hs-00229205_m1; GLUT12: Hs-00376943_m1; β-actin: Hs-99999903_m1) and TaqMan Universal PCR Master Mix (Applied Biosystems). For each PCR assay, 1 µL of cDNA was used, in a final volume of 20 µL containing 1 × TaqMan Universal PCR Master Mix (50 mm KCl, 10 µm EDTA, 10 mm Tris-HCl pH 8.3, and 60 nm Passive Reference) and 1 × (312.5 µm) TaqMan Primer and Probe. Thermocycler conditions were 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Each reaction was done in triplicate and reported values are the mean of each triplicate. For quantification of the real-time PCR, standard curves were constructed from five point dilutions (50, 12.5, 3.13, 0.781, 0.195 ng/µL) of reverse transcribed total RNA. The slopes of the standard curves for GLUTs and β-actin were similar, indicating equal amplification efficiencies. The levels of GLUT and β-actin mRNA were calculated from the threshold cycle number (Ct) during the exponential phase of the PCR amplification. The target GLUT gene mRNA level was normalized by the Ct of β-actin as ΔCt = Ct(GLUT genes) − Ct(β-actin). For relative expression levels, we calculated ΔΔCt as ΔΔCt = ΔCt(tumour) − Ct(normal). A 1-unit decrease in ΔΔCt corresponds to approximately a two-fold higher mRNA level.
The statistical significance of differences in expression levels was determined with the Dunnett’s multiple comparison test, paired t-test or Wilcoxon signed-rank test. The significance level was defined as P < 0.05.
Conventional RT-PCR was used to examine the mRNA expression patterns of GLUT1–14 in normal and tumour kidney tissues. All amplicons were fully sequenced and their identities verified by BLAST sequence similarity analysis against the GLUT mRNA sequences deposited in GenBank. Normal kidney tissue expressed all 14 human GLUT genes, although GLUT7 and GLUT14 were amplified at very low levels in just 34 (83%) and 30 (73%) of 41 samples, respectively. The relatively low expression of GLUT7 and 14 in kidney was confirmed by comparison with the result of amplifications of normal colon and testis (tissues with high mRNA expression of GLUT7 and GLUT14, respectively) [19,20] (Fig. 1). In all the samples from kidney tumours, specific amplicons were produced that corresponded to GLUT1, 3, 6, 8–11 and 13. However, GLUT2, 4, 5 and 12 were amplified only in 25 (83%), 24 (80%) 27 (90%) and 26 (87%) of 30 cases, respectively. The expression levels of GLUT7 and 14 were low in both tumour and normal samples, with no visible difference in expression levels. Consequently we excluded GLUT7 and 14 from the subsequent semiquantitative and real-time PCR experiments.
Semi-quantitative PCR using five-paired samples of clear cell RCC, which is the most common subtype in RCC, indicated that GLUT1, 4, 9 and 12 had the highest relative variation in levels between normal and tumour RNAs (Fig. 2). The band intensity of GLUT1 in all clear cell RCC samples was stronger than in the paired normal sample, whereas the intensities of GLUT4, 9 and 12 amplicons in clear cell RCC samples was always lower than in the paired normal sample. Based on these results, we selected GLUT1-5, 9, 10, and 12 for a comprehensive study of their relative levels in normal vs tumour kidney RNA by real-time quantitative PCR.
Relative mRNA expression levels of the GLUT genes were determined by real-time quantitative PCR of total RNA purified from normal and tumour kidney tissues (clear cell, papillary and chromophobe RCC, and oncocytoma). Mean expression levels are shown in Table 3 and graphically in Fig. 3. Our results indicate that GLUT1 mRNA was present at significantly higher levels in clear cell RCC than in normal tissues (P < 0.001), whereas oncocytoma samples had significantly lower levels than in normal kidney (P < 0.01). GLUT2 was expressed at significantly lower levels in chromophobe RCC and oncocytoma tumours (P < 0.01 and P < 0.001, respectively). There was no significant difference in GLUT3 expression between normal and cancer samples, although there seemed to be an increasing trend in clear cell RCC samples. For GLUT4, clear cell RCC expressed significantly lower levels than normal tissue (P < 0.001). By contrast, chromophobe RCC expressed significantly higher levels than normal tissue (P < 0.05). Although not statistically significant, the expression levels of GLUT4 showed an increasing trend in the oncocytoma samples. Both chromophobe RCC and oncocytoma expressed significantly lower levels of GLUT5 than normal tissue (P < 0.01 and P < 0.001, respectively) whereas there was a statistically non-significant trend towards overexpression in clear cell RCC. GLUT9 was significantly lower in clear cell RCC and oncocytoma (P < 0.01 and P < 0.001, respectively). GLUT10 was expressed at significantly lower levels in oncocytoma (P < 0.001) and with a non-significant trend towards higher levels in papillary RCC. Finally, for GLUT12, both clear cell RCC and papillary RCC expressed significantly lower levels (P < 0.001) than normal tissues. The increasing trend was not significant in chromophobe RCC and oncocytoma samples.
Table 3. GLUT mRNA expression levels in normal and tumour samples evaluated by real-time RT-PCR, data shown as the mean (sd)
Clear cell RCC
N, number of samples; *P < 0.05,†P < 0.01,‡P < 0.001, Dunnett’s multiple comparison test analysis
To exclude possible inter-individual differences, the GLUT1–5, 9, 10 and 12 mRNA expression levels were compared in 11 pairs of clear cell RCC and their corresponding normal tissues from the same patients (Table 4, Fig. 4). GLUT1 mRNA levels were higher in cancer cells than in paired normal cells in all of the cases (P < 0.001). By contrast, in every case GLUT4 and 12 mRNA levels, and in 10 of 11 the GLUT9 mRNA levels were significantly lower in cancer samples (GLUT4, P < 0.001; GLUT9, P < 0.01; GLUT12, P < 0.001) than in normal cells. Neither GLUT2, 3, 5 nor 10 showed any significant difference between cancer cells and the paired normal cells.
Table 4. GLUT mRNA expression levels in 11 clear cell RCC paired samples by real-time RT-PCR, data shown as the mean (sd)
Facilitative GLUTs transport glucose and increased expression of these transporters has been reported in various tumour tissues. GLUT1 [7–13,16] and GLUT3  expression was reported to be higher in various cancers. In addition, it was reported that GLUT4 is elevated in gastric  and ovarian  cancers; GLUT5 in breast  cancer; and GLUT12 in breast  and prostate  cancers. However, few comprehensive studies have been conducted to evaluate the relative expression patterns of GLUT genes in normal and tumour kidney tissues, especially of the more recently identified isoforms . The number of samples that we assessed was necessarily limited, but there were substantial differences in the pattern of GLUT gene expression between normal and tumour tissues, and among histological subtypes of RCCs. GLUT1 mRNA levels were significantly higher in clear cell RCC and GLUT4 mRNA levels were significantly higher in chromophobe RCC. Although not statistically significant, mRNA levels of GLUT3 (clear cell RCC), GLUT4 (oncocytoma), GLUT5 (clear cell RCC), GLUT10 (papillary RCC) and GLUT12 (chromophobe RCC, oncocytoma) tended to be higher.
GLUT1, 3, 4 and 10 have a high affinity for glucose [23,24], while GLUT5  shows a high rate of fructose transport. Although the transport kinetics of GLUT12  have not been elucidated, our results suggest that the high-affinity GLUTs might play a major role in the enhanced glucose uptake shown by kidney tumours. A similar mechanism was suggested based on observations in other types of cancers [11,12].
Nagase et al.  investigated GLUT1 and GLUT4 expression in normal and tumour kidney tissue, using immunohistochemistry, and found strong staining for GLUT1 around the entire periphery of the cells in clear cell RCC. In normal tissue, GLUT1 was only present at the plasma membrane of renal tubule cells; GLUT4 staining was not detected. A mechanism to explain the induction of expression of GLUT1 and GLUT3 in tumours was proposed, involving the von Hippel–Lindau (VHL) gene and the hypoxia-inducible factor α (HIFα) [26–28]. In tumours with mutations in the VHL gene, the defective product is unable to induce the normal degradation of HIFα by ubiquitination . HIFα will then accumulate and induce the expression of hypoxia-responsive genes, such as GLUT1  or GLUT3 . Wiesener et al.  reported that in 40 RCC samples GLUT1 mRNA levels increased concomitantly with the level of HIF-1α in tumour extracts. By contrast, GLUT1 mRNA and protein levels hardly increased in non-clear cell RCC.
The present results show that GLUT1 mRNA expression levels in clear cell RCC are significantly higher than in normal tissue. While not significant, GLUT3 and GLUT5 tended to increase their expression in the clear cell RCC samples. Regarding the other GLUTs, mRNA levels in clear cell RCC for GLUT4, 9 and 12 were significantly lower or essentially unchanged (GLUT2, 6 and 10–13). GLUT7 and 14 were expressed at very low levels. These results suggest that GLUT1 over-expression is the best candidate for enhanced glucose uptake, and GLUT3 and 5 might support enhanced glucose and fructose uptake in clear cell RCC.
There are few reports that GLUT1 mRNA and protein are expressed at low levels in papillary RCC, in contrast to clear cell RCC [8,27,30]. Likewise, GLUT4 protein was not detected in tumour or normal tissue by immunohistochemical staining . In the present study GLUT12 mRNA was significantly lower than in normal tissue. None of the GLUT genes showed a statistically significant increase in expression, although GLUT10 mRNA levels tended to increase. Papillary RCC seems to depend on an increase in GLUT10 for its glucose needs.
For the remaining RCC subtypes, no reports of GLUT expression have been published. Since chromophobe RCC and oncocytoma represent only 5% each of the total RCCs, we had access to a very few samples. GLUT2 and GLUT5 mRNA levels in chromophobe RCC were significantly lower than in normal tissue. By contrast, GLUT4 mRNA in chromophobe RCC was significantly higher. Although there was no significant change, GLUT12 also tended to increase. These results suggest that GLUT4 and GLUT12 are possible candidates for the enhanced glucose uptake in chromophobe RCC. In oncocytoma, GLUT1, 2, 5, 9 and 10 mRNA levels were significantly lower than in normal tissue. Again, although there was no significant change, GLUT4 and GLUT12 tended to increase. Malignant chromophobe RCC, especially the eosinophilic variety, can be difficult to distinguish histologically from the benign oncocytoma. In the present study GLUT expression patterns were similar between chromophobe RCC and oncocytoma, but the difference in GLUT4 mRNA expression level might be useful to distinguish these subtypes.
Importantly, GLUT2, 5, 7 and 9 are partly or exclusively fructose transporters [19,31–33]. mRNA expression levels of GLUT2 and GLUT5 in chromophobe RCC and oncocytoma were significantly lower than in normal tissues. The expression level of GLUT9 in oncocytoma was significantly lower than in normal tissues and tended to decrease in chromophobe RCC. GLUT7 is probably very poorly expressed in both tumour types. Our results suggest that fructose might cease to be used as an energy source by chromophobe RCC and oncocytoma. On the other hand, in clear cell RCC and papillary RCC the mRNA levels of GLUT2, 5 and 9 do not change relative to normal tissue, with the possible exception of GLUT9 in clear cell RCC. These results indicate a clear physiological difference among tumours, even when derived from the same organ. Distinct sources for energy use might represent an important tool in the diagnosis and/or treatment of these tumours.
In summary, we examined the mRNA expression patterns of several GLUT genes in normal and tumour kidney samples. Normal kidney tissue expressed every GLUT mRNA and there were substantial differences in the pattern of GLUT gene expression among histological subtypes of RCC. GLUT1 mRNA levels were significantly increased in clear cell RCC and GLUT4 mRNA levels were significantly increased in chromophobe RCC, suggesting that high-affinity GLUT isoforms might be related to tumour growth. It might be possible to establish novel cancer treatments targeted against these overexpressed transporters.
This study was supported in part by a development grant from Comprehensive Cancer Center of Wake Forest University School of Medicine and used tissues samples from the Comprehensive Cancer Center Tumor Bank.