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Overexpression of GLUT-1 is associated with resistance to radiotherapy and adverse prognosis in squamous cell carcinoma of the oral cavity
Oral Oncology (2007) 43, 796–803
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Overexpression of GLUT-1 is associated with resistance to radiotherapy and adverse prognosis in squamous cell carcinoma of the oral cavity Martin Kunkel a, Maximilian Moergel a, Marcus Stockinger b, Jong-Hyeon Jeong c, Gerhardt Fritz d, Hans-Anton Lehr e, Theresa L. Whiteside f,* a Department of Oral and Maxillofacial Surgery, University of Mainz, Medical Center, Augustusplatz 2, 55131 Mainz, Germany b Department of Radiation Oncology, University of Mainz, Medical Center, Langenbeckstr. 1, 55101 Mainz, Germany c Department of Biostatistics, University of Pittsburgh, Pittsburgh, PA, United States d Institute of Pharmacology and Toxicology, FB 10, University of Giessen, 35392 Giessen, Germany e ´ Vandoise, Rue du Bugnon, Institut Universitaire de Pathologie, Center Hospitalier Universite CH-1011 Lausanne, Switzerland f Departments of Pathology, Immunology and Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, United States
Received 11 August 2006; received in revised form 5 October 2006; accepted 11 October 2006 Available online 4 January 2007
KEYWORDS
Summary This study tested the hypothesis that GLUT-1 is a marker of radioresistance in oral squamous cell carcinomas (OSCC). A GLUT-1 labeling index (LI) was determined by immunohistochemistry in 40 pretreatment OSCC biopsies. Radiation responses were categorized by histopathology of the resection specimens. Associations between the LI and radiation response, Kaplan-Meier survival estimates and Cox regression analysis for the variables GLUT-1, T-stage, N-stage and chemotherapy were examined. The median LI was 64.2% (range 14–100%). Tumors with >65% of GLUT-1 + cells were more resistant to radiation (p = 0.023). Overall survival was higher (p = 0.044) for subjects with low LI (median value). The Cox regression analysis confirmed the prognostic significance of GLUT-1. Our results
Glucose transporter; GLUT-1; Head and neck cancer; Radiation; Radiation resistance
¨ SAK, Deutsch O ¨ sterreichisch SchweiAbbreviations: GLUT-1, Glucose-Transporter 1; CAI-X, Carboanhydrase X; LI, Labeling Index; DO zerischer Arbeitskreis fu ¨r Tumoren im Kiefer-und Gesichtsbereich; OSSC, Oral squamous cell carcinoma; PBS, Phosphate Buered Saline. * Corresponding author. Current address: University of Pittsburgh Cancer Institute, Research Pavilion at the Hillman Cancer Center, Suite 1.27, 5117 Center Avenue, Pittsburgh, PA 15213-1863, United States. Tel.: +1 412 624 0096; fax: +1 412 624 0264. E-mail address: [email protected] (T.L. Whiteside).
1368-8375/$ - see front matter c 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2006.10.009
GLUT-1 is associated with resistance to radiotherapy and adverse prognosis
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indicate that pre-treatment GLUT-1 expression in the tumor is a marker of radioresistance in OSCC, with high expression being associated with poor radiation response and shorter survival. c 2006 Elsevier Ltd. All rights reserved.
Introduction
Materials and methods
Since the landmark studies of Warburg, the glycolytic phenotype has been recognized as a fundamental characteristic of the malignant cell.1 More recently, overexpression of GLUT-1, representing a basic mechanism that may contribute to enhanced glucose metabolism, has been well documented in human solid tumors2–6. Despite slight differences in the staining procedure, type of analysis and cut-off values, all these studies have uniformly associated GLUT-1 overexpression with enhanced tumor aggressiveness and unfavorable clinical outcome. Other studies, including our own, have confirmed the prognostic value of the GLUT-1 expression levels for squamous cell carcinomas of the head and neck.7,8 While the overwhelming clinical evidence has accumulated attesting to the biologic significance of GLUT-1 in solid tumors, the metabolic consequences of increased glucose transport are still not completely understood. In particular, the mechanisms relating increased GLUT-1 expression to tumor aggressiveness and poor survival have remained largely unexplored. It has been suggested that like CAI-X and HIF 1a, GLUT-1 might represent an intrinsic marker of hypoxia.6,9 However, other studies have identified GLUT-1 overexpression as an early event after Ras and Src transformation,10 and, hence, not primarily related to hypoxia. Consistent with these latter findings, Pedersen and coworkers more recently reported a hypoxia-independent effect of GLUT-1 on radiation resistance in small-cell lung cancer cells.11 Recent evidence also emphasizes a fundamental role of glucose availability in the regulation of cell survival and apoptosis.12,13 Taken together, these findings give raise to the hypothesis that the prognostic significance of GLUT-1 expression might, at least in part, be related to the influence of glucose metabolism on radiation-induced tumor cell apoptosis. To the best of our knowledge, the potential relationship between GLUT-1 expression in tumor cells and the tumor response to radiotherapy has not been systematically analyzed in oral squamous cell carcinomas (OSSC). Therefore, in the present study we have set out to investigate whether GLUT-1 expression is related to the radioresistance of tumors at a clinically relevant level. We have compared the GLUT-1 expression in pre-therapeutic biopsies of OSCC with radiation effects, as measured by the standardized histological workup of the post-radiotherapy resection specimens. We have also related GLUT-1 expression to clinical outcome in a largely homogeneous population of oral cancer patients. Our results establish GLUT-1 as a marker of radioresistance in OSCC, with the high GLUT-1 expression being associated with poor radiation response as well as with an unfavorable clinical outcome.
Patients and treatment Forty patients (7 female and 33 male), age 34–72 years (mean 52) were enrolled in this retrospective study. The criteria for the inclusion in this analysis were as follows: diagnosis of primary OSCC; preoperative radiotherapy (36 Gy) followed by surgical resection with curative intention; follow-up data available; antigen preservation in the biopsy tissues confirmed by internal vimentin control.14 Retrieval of paraffin-embedded biopsy material for the study was approved by the institutional review board of the University of Mainz/Landesaerztekammer Rheinland Pfalz. The patients were treated at the Department of Oral and Maxillofacial Surgery at the University of Mainz Medical Center according to the ‘‘Essen’’ protocol:15 preoperative radiation therapy (36 Gy) followed by radical surgical resection with a safety margin of at least 1 cm. Lymph node resection ¨ SAK cooperative followed the recommendations of the DO ¨ sterreichisch Schweizerischer Arbeitskreis group (Deutsch O fu ¨r Tumoren im Kiefer- und Gesichtsbereich): The N0-neck was treated by a selective anterolateral neck dissection (Level I/II/III) when the primary tumor was located in the anterior or lateral oral cavity. When intraoperative frozen section examination of suspicious lymph nodes revealed metastatic spread, the neck dissection was extended to Level IV and V. Tumors located in posterior (retromolar) sites were a priori treated by a complete lymph node dissection (Levels I–V). Although the concept of an induction radiotherapy according to the ‘‘Essen’’ protocol has largely been abandoned for oral squamous cell carcinomas, this cohort recruited from 1995 to 2000 proved highly valuable for studies addressing the issue of radiation response, since the complete resection specimens allow an objective measurement of radiation effects. In our study population, the sites of the primary tumors were as follows: 3 tumors of the maxilla including the palate; 16 tumors in the floor of the mouth; 10 tumors in the tongue; 8 tumors in the gingiva of the mandible and the retromolar trigone and 4 in other locations. The TNM staging categories were determined according to the criteria established by the AJCC/UICC.16 Stage grouping of the patients was as follows: Stage I: 1 patient; Stage II: 5 patients; Stage III: 4 patients; Stage IV: 30 patients.
Radiation protocol After histological confirmation of the malignancy, radiotherapy treatment was planned based on CT sections using
798 computerized planning systems (Helax-TMS, Nucleotron B.V., Ax Veenendaal, Netherlands or OSS, Royal Philips Electronics, Eindhoven, Netherlands). External radiotherapy was delivered using a cobalt-60 machine (Gammatron 3, Siemens AG, Munich, Germany) or/and linear accelerators (Type KD-2 or type Mevatron-77, both manufactured by Siemens AG, Munich, Germany). To ensure proper patient positioning with every radiation session, ink markings were drawn on the skin. Preoperative total dose of 36 Gy was administered by bilateral opposing beams (1.3 MV photons), covering tumor site and bilateral draining lymphatics of the neck, including the supraclavicular region. The dose was given in fractions of 2 Gy/day through 5 days/week. When additional postoperative radiation was to be expected based on the initial clinical findings or the imaging results, spinal marrow was blocked after 30 Gy of preoperative therapy and bilateral opposing 10 MeV electron beams were attached to the dorsal margin of blocked photon beams. In cases of incomplete resection (positive resection margins on histopathologic examination), lymphangiosis carcinomatosa or extracapsular spread of involved nodes, additional postoperative radiotherapy was given up to a complete dose of 60 Gy in fractions of 2 Gy/day through 5 days/week. Radiation response to 36 Gy administered preoperatively was categorized based on the histologic examination of resection specimens. As this classification does not address the surgical resection status but instead addresses the response to radiation therapy, it is designated Rad-R(0–2), which more accurately describes attributes of the residual tumor after radiation. In case of a minimal or partial response (bulky residual tumor on clinical and pathological evaluations), the specimens were classified as ‘‘Rad-R2’’. In case of a complete clinical response, the specimens were further subdivided according to the histological findings into ‘‘Rad-R1’’ (remaining viable tumor cells on histological evaluation) and ‘‘Rad-R0’’ (no residual vital tumor on histological evaluation). At the time of survival analysis, 18 patients had died, 3 of causes unrelated to their tumors. The follow-up period for the surviving patients ranged from 25 to 106 months (median 62 months).
Immunohistochemistry Specimens and staining For immunohistochemical evaluation, paraffin blocks of the formalin-fixed biopsies were retrieved from the pathology archives. In addition to tumor biopsies of patients included in this analysis, 118 blocks of non-irradiated OSCC were also obtained. In every specimen, the quality of antigen preservation was assessed by immunostaining for vimentin.14 Sections were immunostained for GLUT-1 using an avidin– biotin technique according to the standard protocol. Briefly, 6 lm sections were deparaffinazed and rehydrated. For antigen retrieval, the sections were exposed to hot steam for 20 min in 1 mM EDTA buffer (pH 8.0). After cooling to room temperature, sections were quenched for endogenous peroxidase activity by 0.1% H2O2 treatment (20 min) and then incubated with 10% normal goat serum (30 min) to block nonspecific binding of immunoglobulins to the tissue, followed by incubation with the polyclonal rabbit anti-hu-
M. Kunkel et al. man GLUT-1-antibody (MYM AB 1351; Chemicon, Temecula, CA, USA) at a final dilution of 1:1000 (60 min, room temperature). The LSAB II Kit (DAKO Diagnostics, Hamburg Germany) was used for color development with peroxidase and 3,3-diamino benzidine (DAB). Nuclei were lightly counterstained with hematoxylin. Negative controls were obtained by omitting the primary antibody. Erythrocytes, which were present in every section, served as internal controls for GLUT-1 to assure constant immunostaining intensity. For the assessment of GLUT-1 expression at the borderline of the tumors, simultaneous GLUT-1 and KI67 staining was performed in 66 Blocks of non-irradiated OSCC using anti Ki-67 and APAAP methodology. After the GLUT-1 staining as described above, the sections were flushed extensively with PBS and incubated with the next primary antibody (anti-human Ki-67 mAb, clone MIB-1, DIANOVA, Hamburg, Germany) at a final dilution 1:30 for 60 min at room temperature. For visualisation Goat anti-mouse Ab (Z420) at a final dilution of 1:25, APAAP mouse complex (D651) at a final dilution of 1:50 and BCIP/NBT substrate (K598) were used (all from DAKO Diagnostics, Hamburg Germany). Evaluation of stained sections GLUT-1 immunostaining was evaluated by the same investigator (MM) blinded to the clinical and follow-up data. For quantitative evaluation, a GLUT-1 Labeling Index (LI) was established for each tumor, based on the percentage of tumor cells that expressed the protein. Briefly, a grid of 100 squares (0.025 · 0.025 mm each) was projected into the field of view of the microscope. Ten randomly selected high power fields (magnification 400·) were photographed using a Leica DM/RBE microscope equipped with a digital camera (Leica Wild MPS52) and an image system (IM50 image manager V 1.2). Using the grid, the total number of tumor cells and the number of GLUT-1 stained cells was determined for each 10 · 10 matrix in the high power field. The tumor cells obtained in 1000 squares were counted for each specimen. We distinguished tumors with a low LI (the number of GLUT-1 + tumor cells was < the median value defined for all tumors) from these with a high LI (the number of GLUT-1 + tumor cells was > the median value defined for all tumors).
Statistical analysis The analysis of the association between GLUT-1 LI and the radiation response (groups Rad-R0, Rad-R1 and Rad-R2 based on the histopathological evaluation of the residual tumor specimens) utilized the Fisher’s exact test. The LI values were compared between clinical responders (groups Rad-R0 + Rad-R1) and incomplete responders (group RadR2), using Wilcoxon’s rank sum test or the Mann and Whitney test and among all groups (Rad-R0, -R1 and -R2), using the non-parametric Kruskal–Wallis test. The reported p-values are two-sided. Survival probabilities were estimated by the Kaplan–Meier method.17 The endpoint of interest was time from tumor resection to tumor-related death. Three cases of death from other causes than tumor were coded as censored
GLUT-1 is associated with resistance to radiotherapy and adverse prognosis observations. The patients’ data were first dichotomized by the median of the GLUT-1 expression to evaluate association with the survival outcome. The log-rank test was used to obtain p-values for two-group comparisons without adjustment for other potential prognostic factors. Multiple regression analysis was performed by using the proportional hazards model18 with the following dichotomized co-variates as independent variables: GLUT-1 LI (low vs. high), T-stage (T1 + T2 vs. T3 + T4), N-stage (N0 vs.N > 0), chemotherapy (administered vs. not administered), and grading (G1 + G2 vs. G3). Two sided p-values were obtained from the Wald-tests for testing any statistical significance of the regression coefficients.
Results GLUT-1 expression in OSCC Expression of GLUT-1 was positive in all OSCC specimens examined as part of this study. To more completely evaluate GLUT-1 expression in OSCC, specimens of non-irradiated tumors (not included in this analysis) were also examined. The LI was found to be highly variable among tumors, ranging from 14% to 100%. Representative examples of tumors showing ‘‘high’’ and ‘‘low’’ GLUT-1 expression are given in Figure 1. Various staining patterns of GLUT-1 were observed within individual tumors, including either membraneous or cytoplasmic staining or a combination of the two. However, the type of staining pattern was not associated with radiation response or the clinical outcome. GLUT-1 expression could be very intense not only inside the tumor mass but also at the borderline in some tumors,
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as shown in Figure 2. Among 66 blocks originally sectioned, 39 contained non-irradiated tumor with the characteristic epithelial borderline when stained for GLUT-1 and Ki-67. In 23 out of these 39 specimens, a well demarcated GLUT1 positive border was noticed within the squamous epithelium (see Fig. 2). The remaining specimens showed either an ill defined tumor borderline, a continuous increase of GLUT-1 staining along with the increasing grade of dysplasia around the tumors or, rarely, weak staining for GLUT-1.
Association between GLUT-1 expression and radiation resistance When a preoperative radiation dose of 36 Gy had been delivered, 23 tumors showed only minimal or partial shrinkage, and these specimens were classified as Rad-R2 based on the macroscopically detected presence of residual tumors. Complete clinical response was noticed in 17 tumors. Upon histopathological evaluation of these cases, 11 specimens were classified as Rad-R1 (positive for residual tumor cells on histological assessment) and 6 specimens were classified as Rad-R0 (no residual tumor cells on histological assessment). The average LI was 53.7% (±17) in the group RadR0, 54.3% (±16) in the group Rad-R1 and 70% (±16) in the group Rad-R2 (p = 0.0349). Clinical responders (groups Rad-R0 + Rad-R1) showed a significantly lower expression of GLUT-1 when compared to the incomplete responders classified as Rad-R2 (p = 0.009). By setting a cutoff point of 65% for the GLUT-1 expression, and using the Fisher’s exact test, a significant association (p = 0.023) was observed between the GLUT-1 LI and the resistance of tumor cells to radiation.
Figure 1 Examples of GLUT-1 expression in OSCC as determined by immuno-histochemistry. The paraffin sections demonstrate GLUT-1 staining in the biopsy material of the tumors belonging to: (a) the low LI subgroup (
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Figure 2 Co-expression of GLUT-1 and KI-67 at the outer border of an OSSC. Note that the intense pathologic expression of GLUT-1 demarcates the tumor (b) from the adjacent tissues. Immunohistochemistry of the resection specimen of a non-irradiated tumor. Original magnification, 200·.
Association between GLUT-1 expression in the tumor and patient survival During the follow-up period, 4 of 20 patients (20%) in the ‘‘low LI’’ group (LI < median) and 11 of 20 patients (55%) in the ‘‘high LI’’ group (LI P median) had died. The difference in survival between the two groups was statistically significant (p = 0.044). The Kaplan–Meier plot in Figure 4 suggests that higher GLUT-1 LI predicts a lower survival probability in patients undergoing preoperative radiation therapy. When considered as a single continuous variable in the Cox regression model, GLUT-1 was assigned a hazard ratio of 1.028 per one unit increase of LI. The dichotomized GLUT-1 LI was also confirmed as a significant prognostic marker in a proportional hazards regres-
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GLUT-1 LT [%]
The association between GLUT-1 expression and radiation resistance was also significant for the more homogeneous cohort of 29 locally advanced (T3 and T4) tumors (Figure 3). In this subpopulation, the average LI was 42.7% (±18) for the Rad-R0 group, 53.9% (±17) for the Rad-R1 group and 71.8% (±16) for the Rad-R2 group (p = 0.0116). The difference between clinical responders (groups Rad-R0 + RadR1) and incomplete responders (group Rad-R2) was highly significant (p = 0.004). In addition, the Fisher’s exact test confirmed a significant association between GLUT-1 expression in the tumor and radiation resistance (p = 0.032). These results suggest that GLUT-1 expression could be considered as a marker of radioresistance in OSCC, with a high GLUT-1 expression being associated with poor radiation response and vice versa.
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40
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group Rad-R0
group Rad-R1
group Rad-R2
Figure 3 GLUT-1 expression stratified according to radiation responses in the homogenous group of 29 OSCC patients with T3 and T4 tumors. Resistance to radiation was associated with an increased GLUT-1 labeling index and vice versa (p = 0.023). The three groups were defined on the basis of histological evaluations as: Group Rad-R0 (n = 3): no residual vital tumor; Group Rad-R1 (n = 9): remaining viable tumor cells; Group Rad-R2 (n = 17): bulky residual tumor (clinical and pathological evaluation). The box plots show median % of LI (black bar); the boxes are interquartile (25–75%) ranges, and the whiskers extend to the maximum and minimum values.
sion analysis, controlled for T-stage, N-stage, grading, and administration of chemotherapy (Cisplatin). The estimated hazard ratio between low and high GLUT-1 LI was 5.11 with
GLUT-1 is associated with resistance to radiotherapy and adverse prognosis 1.0
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LI < median (n=20)
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1.4
LI > median (n=20)
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0.0 0
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Time [months] Figure 4 Kaplan–Meier survival estimates generated by univariate analysis for high LI (>median) vs. low LI (
a p-value of 0.035. The hazard ratio, estimates along with 95% confidence intervals and p-values from the Wald tests for all variables are given in Table 1. The data indicate that the GLUT-1 LI could be considered a negative predictive marker of prognosis in preoperatively irradiated OSCC, with a high GLUT-1 LI being associated with an unfavorable outcome. Both the impact of GLUT-1 expression on radiation response and the association between GLUT-1 expression and survival suggest a biologically and clinically relevant influence of glucose metabolism on radiation responses in OSCC.
Discussion The role of hypoxia as a potential factor counteracting the efficacy of radiotherapy has been thoroughly investigated in the last decade.19–21 These studies have established a significant correlation between endogenous markers of hypoxia and clinical outcome.22–24 Only recently, Koukourakis and coworkers have reported a strong association between two
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separate ‘‘hypoxia markers’’ (HIF-2A, CA9) and survival in a large cohort of head and neck cancer patients originally recruited in the CHART-randomized trial.25 There are two fundamental drawbacks when outcome effects are directly attributed to the hypoxia-induced radiation failure. First, the so called ‘‘endogenous markers of hypoxia’’, including HIF, CA9 and GLUT-1, do not exclusively correspond to the tumor oxygenation status. Rather, expression in tumor cells of these markers might reflect the activity of other oncogenic pathways independent of hypoxia. In fact, two recent reports comparing oxygen tension in cervical cancers to HIF-1a and GLUT-1 expression provided strong evidence for a regulation of these pathways independently of hypoxia.26–28 Further Chen et al.28 have identified a hypoxia-independent promotive effect of Ras on the GLUT-1 expression triggered via the HIF-1a binding site in the GLUT-1 promoter region. These results are in agreement with the more recent report by Elstrom and coworkers on hypoxia-independent stimulation of glycolysis by Akt transfection.29 In our hands, a hypoxia-independent up-regulation of GLUT-1 expression is also supported by the characteristic locoregional distribution pattern of staining for GLUT-1 seen in 23/39 tumors. The distinct localization of GLUT-1 staining to the tumor border (see Fig. 2) cannot be explained by a gradient of oxygen saturation between the tumor and the immediate proximate tissue but rather suggests hypoxia-independent overexpression of GLUT-1. The second drawback is a methodological one. The ‘‘true’’ efficacy of radiation therapy has not been verified in previous studies, because no complete or even representative histological controls were performed. The contribution of the cellular markers to survival was exclusively attributed to radiation resistance, although several other biological tumor properties (i.e., tumor cell chemosensitivity or tumor invasiveness) could also have contributed to the clinical outcome. In order to go a step further, our study examined two clinical endpoints with regard to the enhanced expression of GLUT-1 in OSCC: (i) radiation response measured by histological evaluation of complete resection specimens as first-hand information and (ii) overall survival as a confirmative parameter. In our investigation, a consistent and significant influence of GLUT-1 could be verified for both endpoints. The GLUT-1 expression level was inversely correlated to radiation response and, most important, it was predictive of the clinical outcome in both univariate and
Table 1 Survival analysis based on GLUT-1 overexpression in the tumor and controlled for other prognostically important variables in OSCCa Variable
Definition of groups (Group 1 vs. Group 2)
95% Confidence interval for the hazard ratio
Hazard ratio (Group 2 relative to Group 1)
p-value (Wald)
T-stage N-stage Grade of differentiation Chemotherapy (Cisplatin)
T1 + T2 vs. T3 + T4 N0 vs. N > 0 G1 + 2 vs. G3 Administered vs. not administered
0.66–32.21 0.34–24.42 0.13–2.69 2.49–77.15
4.61 2.88 0.60 13.85
0.12 0.33 0.50 0.003
1.12–23.39
5.12
0.035
GLUT-1 (LI) a
Cox regression analysis.
802 multivariate survival analyses, with a high GLUT-1 expression being associated with poor survival. Although the association between GLUT-1 expression and radioresistance has not been established at the clinical level in other human solid tumors to date, in vitro experiments and tumor xenograft studies reported by Pedersen and coworkers argue that GLUT-1 expression plays a hypoxia-independent role in the modulation of radiation susceptibility.11 These investigators demonstrated a linkage between GLUT-1 expression and radiation resistance in two cell sublines (CPH-54A and CPH-54B) derived from a single small cell carcinoma of the lung. Apart from tumor metabolism, the protective effects of glucose transporters and glucose consumption are well known in cardiology.30 Several lines of evidence support the concept that glucose-dependent inhibition of the mitochondrial apoptotic pathway is highly relevant to the protection of cardiac myocytes against apoptosis.30 Additional potential protective mechanisms involving glucose include the cellular redox control by NADH supply31 and caspase-2 inhibition by NADPH generation.13 It is intriguing to note, that radiation-induced cell death is, to a substantial extent, mediated by the same pathways,32 providing support for the hypothesis that cellular glucose metabolism directly affects cellular radiosensitivity. Taken together, our comparative analysis of GLUT-1 expression and therapeutic efficiency of radiation, as measured by histology and overall survival, provide first clinical evidence that GLUT-1 may be a useful parameter for identifying patients with radioresistant OSCC, which could not be expected to be cured by radiotherapy alone. Furthermore, our results confirm the value of GLUT-1 expression as a predictive marker for this tumor type. In addition to providing prognostic information, our data suggest that modulation of radiation resistance by inhibition of glucose transport in the tumor may be a novel strategy to improve effectiveness of radiotherapy in OSCC.
Conflict of interest None declared.
Acknowledgements This work was supported in part by a grant of the ‘‘Mainzer Forschungsfo ¨rderungsprogramm des Fachbereichs Medizin’’ to M. Kunkel (MAIFOR: Glukosetransporter) and by the NIH grant PO1-DE12321 to T.L. Whiteside.
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Overexpression of GLUT-1 is associated with resistance to radiotherapy and adverse prognosis in squamous cell carcinoma of the oral cavity Martin Kunkel a, Maximilian Moergel a, Marcus Stockinger b, Jong-Hyeon Jeong c, Gerhardt Fritz d, Hans-Anton Lehr e, Theresa L. Whiteside f,* a Department of Oral and Maxillofacial Surgery, University of Mainz, Medical Center, Augustusplatz 2, 55131 Mainz, Germany b Department of Radiation Oncology, University of Mainz, Medical Center, Langenbeckstr. 1, 55101 Mainz, Germany c Department of Biostatistics, University of Pittsburgh, Pittsburgh, PA, United States d Institute of Pharmacology and Toxicology, FB 10, University of Giessen, 35392 Giessen, Germany e ´ Vandoise, Rue du Bugnon, Institut Universitaire de Pathologie, Center Hospitalier Universite CH-1011 Lausanne, Switzerland f Departments of Pathology, Immunology and Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, United States
Received 11 August 2006; received in revised form 5 October 2006; accepted 11 October 2006 Available online 4 January 2007
KEYWORDS
Summary This study tested the hypothesis that GLUT-1 is a marker of radioresistance in oral squamous cell carcinomas (OSCC). A GLUT-1 labeling index (LI) was determined by immunohistochemistry in 40 pretreatment OSCC biopsies. Radiation responses were categorized by histopathology of the resection specimens. Associations between the LI and radiation response, Kaplan-Meier survival estimates and Cox regression analysis for the variables GLUT-1, T-stage, N-stage and chemotherapy were examined. The median LI was 64.2% (range 14–100%). Tumors with >65% of GLUT-1 + cells were more resistant to radiation (p = 0.023). Overall survival was higher (p = 0.044) for subjects with low LI (
Glucose transporter; GLUT-1; Head and neck cancer; Radiation; Radiation resistance
¨ SAK, Deutsch O ¨ sterreichisch SchweiAbbreviations: GLUT-1, Glucose-Transporter 1; CAI-X, Carboanhydrase X; LI, Labeling Index; DO zerischer Arbeitskreis fu ¨r Tumoren im Kiefer-und Gesichtsbereich; OSSC, Oral squamous cell carcinoma; PBS, Phosphate Buered Saline. * Corresponding author. Current address: University of Pittsburgh Cancer Institute, Research Pavilion at the Hillman Cancer Center, Suite 1.27, 5117 Center Avenue, Pittsburgh, PA 15213-1863, United States. Tel.: +1 412 624 0096; fax: +1 412 624 0264. E-mail address: [email protected] (T.L. Whiteside).
1368-8375/$ - see front matter c 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2006.10.009
GLUT-1 is associated with resistance to radiotherapy and adverse prognosis
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indicate that pre-treatment GLUT-1 expression in the tumor is a marker of radioresistance in OSCC, with high expression being associated with poor radiation response and shorter survival. c 2006 Elsevier Ltd. All rights reserved.
Introduction
Materials and methods
Since the landmark studies of Warburg, the glycolytic phenotype has been recognized as a fundamental characteristic of the malignant cell.1 More recently, overexpression of GLUT-1, representing a basic mechanism that may contribute to enhanced glucose metabolism, has been well documented in human solid tumors2–6. Despite slight differences in the staining procedure, type of analysis and cut-off values, all these studies have uniformly associated GLUT-1 overexpression with enhanced tumor aggressiveness and unfavorable clinical outcome. Other studies, including our own, have confirmed the prognostic value of the GLUT-1 expression levels for squamous cell carcinomas of the head and neck.7,8 While the overwhelming clinical evidence has accumulated attesting to the biologic significance of GLUT-1 in solid tumors, the metabolic consequences of increased glucose transport are still not completely understood. In particular, the mechanisms relating increased GLUT-1 expression to tumor aggressiveness and poor survival have remained largely unexplored. It has been suggested that like CAI-X and HIF 1a, GLUT-1 might represent an intrinsic marker of hypoxia.6,9 However, other studies have identified GLUT-1 overexpression as an early event after Ras and Src transformation,10 and, hence, not primarily related to hypoxia. Consistent with these latter findings, Pedersen and coworkers more recently reported a hypoxia-independent effect of GLUT-1 on radiation resistance in small-cell lung cancer cells.11 Recent evidence also emphasizes a fundamental role of glucose availability in the regulation of cell survival and apoptosis.12,13 Taken together, these findings give raise to the hypothesis that the prognostic significance of GLUT-1 expression might, at least in part, be related to the influence of glucose metabolism on radiation-induced tumor cell apoptosis. To the best of our knowledge, the potential relationship between GLUT-1 expression in tumor cells and the tumor response to radiotherapy has not been systematically analyzed in oral squamous cell carcinomas (OSSC). Therefore, in the present study we have set out to investigate whether GLUT-1 expression is related to the radioresistance of tumors at a clinically relevant level. We have compared the GLUT-1 expression in pre-therapeutic biopsies of OSCC with radiation effects, as measured by the standardized histological workup of the post-radiotherapy resection specimens. We have also related GLUT-1 expression to clinical outcome in a largely homogeneous population of oral cancer patients. Our results establish GLUT-1 as a marker of radioresistance in OSCC, with the high GLUT-1 expression being associated with poor radiation response as well as with an unfavorable clinical outcome.
Patients and treatment Forty patients (7 female and 33 male), age 34–72 years (mean 52) were enrolled in this retrospective study. The criteria for the inclusion in this analysis were as follows: diagnosis of primary OSCC; preoperative radiotherapy (36 Gy) followed by surgical resection with curative intention; follow-up data available; antigen preservation in the biopsy tissues confirmed by internal vimentin control.14 Retrieval of paraffin-embedded biopsy material for the study was approved by the institutional review board of the University of Mainz/Landesaerztekammer Rheinland Pfalz. The patients were treated at the Department of Oral and Maxillofacial Surgery at the University of Mainz Medical Center according to the ‘‘Essen’’ protocol:15 preoperative radiation therapy (36 Gy) followed by radical surgical resection with a safety margin of at least 1 cm. Lymph node resection ¨ SAK cooperative followed the recommendations of the DO ¨ sterreichisch Schweizerischer Arbeitskreis group (Deutsch O fu ¨r Tumoren im Kiefer- und Gesichtsbereich): The N0-neck was treated by a selective anterolateral neck dissection (Level I/II/III) when the primary tumor was located in the anterior or lateral oral cavity. When intraoperative frozen section examination of suspicious lymph nodes revealed metastatic spread, the neck dissection was extended to Level IV and V. Tumors located in posterior (retromolar) sites were a priori treated by a complete lymph node dissection (Levels I–V). Although the concept of an induction radiotherapy according to the ‘‘Essen’’ protocol has largely been abandoned for oral squamous cell carcinomas, this cohort recruited from 1995 to 2000 proved highly valuable for studies addressing the issue of radiation response, since the complete resection specimens allow an objective measurement of radiation effects. In our study population, the sites of the primary tumors were as follows: 3 tumors of the maxilla including the palate; 16 tumors in the floor of the mouth; 10 tumors in the tongue; 8 tumors in the gingiva of the mandible and the retromolar trigone and 4 in other locations. The TNM staging categories were determined according to the criteria established by the AJCC/UICC.16 Stage grouping of the patients was as follows: Stage I: 1 patient; Stage II: 5 patients; Stage III: 4 patients; Stage IV: 30 patients.
Radiation protocol After histological confirmation of the malignancy, radiotherapy treatment was planned based on CT sections using
798 computerized planning systems (Helax-TMS, Nucleotron B.V., Ax Veenendaal, Netherlands or OSS, Royal Philips Electronics, Eindhoven, Netherlands). External radiotherapy was delivered using a cobalt-60 machine (Gammatron 3, Siemens AG, Munich, Germany) or/and linear accelerators (Type KD-2 or type Mevatron-77, both manufactured by Siemens AG, Munich, Germany). To ensure proper patient positioning with every radiation session, ink markings were drawn on the skin. Preoperative total dose of 36 Gy was administered by bilateral opposing beams (1.3 MV photons), covering tumor site and bilateral draining lymphatics of the neck, including the supraclavicular region. The dose was given in fractions of 2 Gy/day through 5 days/week. When additional postoperative radiation was to be expected based on the initial clinical findings or the imaging results, spinal marrow was blocked after 30 Gy of preoperative therapy and bilateral opposing 10 MeV electron beams were attached to the dorsal margin of blocked photon beams. In cases of incomplete resection (positive resection margins on histopathologic examination), lymphangiosis carcinomatosa or extracapsular spread of involved nodes, additional postoperative radiotherapy was given up to a complete dose of 60 Gy in fractions of 2 Gy/day through 5 days/week. Radiation response to 36 Gy administered preoperatively was categorized based on the histologic examination of resection specimens. As this classification does not address the surgical resection status but instead addresses the response to radiation therapy, it is designated Rad-R(0–2), which more accurately describes attributes of the residual tumor after radiation. In case of a minimal or partial response (bulky residual tumor on clinical and pathological evaluations), the specimens were classified as ‘‘Rad-R2’’. In case of a complete clinical response, the specimens were further subdivided according to the histological findings into ‘‘Rad-R1’’ (remaining viable tumor cells on histological evaluation) and ‘‘Rad-R0’’ (no residual vital tumor on histological evaluation). At the time of survival analysis, 18 patients had died, 3 of causes unrelated to their tumors. The follow-up period for the surviving patients ranged from 25 to 106 months (median 62 months).
Immunohistochemistry Specimens and staining For immunohistochemical evaluation, paraffin blocks of the formalin-fixed biopsies were retrieved from the pathology archives. In addition to tumor biopsies of patients included in this analysis, 118 blocks of non-irradiated OSCC were also obtained. In every specimen, the quality of antigen preservation was assessed by immunostaining for vimentin.14 Sections were immunostained for GLUT-1 using an avidin– biotin technique according to the standard protocol. Briefly, 6 lm sections were deparaffinazed and rehydrated. For antigen retrieval, the sections were exposed to hot steam for 20 min in 1 mM EDTA buffer (pH 8.0). After cooling to room temperature, sections were quenched for endogenous peroxidase activity by 0.1% H2O2 treatment (20 min) and then incubated with 10% normal goat serum (30 min) to block nonspecific binding of immunoglobulins to the tissue, followed by incubation with the polyclonal rabbit anti-hu-
M. Kunkel et al. man GLUT-1-antibody (MYM AB 1351; Chemicon, Temecula, CA, USA) at a final dilution of 1:1000 (60 min, room temperature). The LSAB II Kit (DAKO Diagnostics, Hamburg Germany) was used for color development with peroxidase and 3,3-diamino benzidine (DAB). Nuclei were lightly counterstained with hematoxylin. Negative controls were obtained by omitting the primary antibody. Erythrocytes, which were present in every section, served as internal controls for GLUT-1 to assure constant immunostaining intensity. For the assessment of GLUT-1 expression at the borderline of the tumors, simultaneous GLUT-1 and KI67 staining was performed in 66 Blocks of non-irradiated OSCC using anti Ki-67 and APAAP methodology. After the GLUT-1 staining as described above, the sections were flushed extensively with PBS and incubated with the next primary antibody (anti-human Ki-67 mAb, clone MIB-1, DIANOVA, Hamburg, Germany) at a final dilution 1:30 for 60 min at room temperature. For visualisation Goat anti-mouse Ab (Z420) at a final dilution of 1:25, APAAP mouse complex (D651) at a final dilution of 1:50 and BCIP/NBT substrate (K598) were used (all from DAKO Diagnostics, Hamburg Germany). Evaluation of stained sections GLUT-1 immunostaining was evaluated by the same investigator (MM) blinded to the clinical and follow-up data. For quantitative evaluation, a GLUT-1 Labeling Index (LI) was established for each tumor, based on the percentage of tumor cells that expressed the protein. Briefly, a grid of 100 squares (0.025 · 0.025 mm each) was projected into the field of view of the microscope. Ten randomly selected high power fields (magnification 400·) were photographed using a Leica DM/RBE microscope equipped with a digital camera (Leica Wild MPS52) and an image system (IM50 image manager V 1.2). Using the grid, the total number of tumor cells and the number of GLUT-1 stained cells was determined for each 10 · 10 matrix in the high power field. The tumor cells obtained in 1000 squares were counted for each specimen. We distinguished tumors with a low LI (the number of GLUT-1 + tumor cells was < the median value defined for all tumors) from these with a high LI (the number of GLUT-1 + tumor cells was > the median value defined for all tumors).
Statistical analysis The analysis of the association between GLUT-1 LI and the radiation response (groups Rad-R0, Rad-R1 and Rad-R2 based on the histopathological evaluation of the residual tumor specimens) utilized the Fisher’s exact test. The LI values were compared between clinical responders (groups Rad-R0 + Rad-R1) and incomplete responders (group RadR2), using Wilcoxon’s rank sum test or the Mann and Whitney test and among all groups (Rad-R0, -R1 and -R2), using the non-parametric Kruskal–Wallis test. The reported p-values are two-sided. Survival probabilities were estimated by the Kaplan–Meier method.17 The endpoint of interest was time from tumor resection to tumor-related death. Three cases of death from other causes than tumor were coded as censored
GLUT-1 is associated with resistance to radiotherapy and adverse prognosis observations. The patients’ data were first dichotomized by the median of the GLUT-1 expression to evaluate association with the survival outcome. The log-rank test was used to obtain p-values for two-group comparisons without adjustment for other potential prognostic factors. Multiple regression analysis was performed by using the proportional hazards model18 with the following dichotomized co-variates as independent variables: GLUT-1 LI (low vs. high), T-stage (T1 + T2 vs. T3 + T4), N-stage (N0 vs.N > 0), chemotherapy (administered vs. not administered), and grading (G1 + G2 vs. G3). Two sided p-values were obtained from the Wald-tests for testing any statistical significance of the regression coefficients.
Results GLUT-1 expression in OSCC Expression of GLUT-1 was positive in all OSCC specimens examined as part of this study. To more completely evaluate GLUT-1 expression in OSCC, specimens of non-irradiated tumors (not included in this analysis) were also examined. The LI was found to be highly variable among tumors, ranging from 14% to 100%. Representative examples of tumors showing ‘‘high’’ and ‘‘low’’ GLUT-1 expression are given in Figure 1. Various staining patterns of GLUT-1 were observed within individual tumors, including either membraneous or cytoplasmic staining or a combination of the two. However, the type of staining pattern was not associated with radiation response or the clinical outcome. GLUT-1 expression could be very intense not only inside the tumor mass but also at the borderline in some tumors,
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as shown in Figure 2. Among 66 blocks originally sectioned, 39 contained non-irradiated tumor with the characteristic epithelial borderline when stained for GLUT-1 and Ki-67. In 23 out of these 39 specimens, a well demarcated GLUT1 positive border was noticed within the squamous epithelium (see Fig. 2). The remaining specimens showed either an ill defined tumor borderline, a continuous increase of GLUT-1 staining along with the increasing grade of dysplasia around the tumors or, rarely, weak staining for GLUT-1.
Association between GLUT-1 expression and radiation resistance When a preoperative radiation dose of 36 Gy had been delivered, 23 tumors showed only minimal or partial shrinkage, and these specimens were classified as Rad-R2 based on the macroscopically detected presence of residual tumors. Complete clinical response was noticed in 17 tumors. Upon histopathological evaluation of these cases, 11 specimens were classified as Rad-R1 (positive for residual tumor cells on histological assessment) and 6 specimens were classified as Rad-R0 (no residual tumor cells on histological assessment). The average LI was 53.7% (±17) in the group RadR0, 54.3% (±16) in the group Rad-R1 and 70% (±16) in the group Rad-R2 (p = 0.0349). Clinical responders (groups Rad-R0 + Rad-R1) showed a significantly lower expression of GLUT-1 when compared to the incomplete responders classified as Rad-R2 (p = 0.009). By setting a cutoff point of 65% for the GLUT-1 expression, and using the Fisher’s exact test, a significant association (p = 0.023) was observed between the GLUT-1 LI and the resistance of tumor cells to radiation.
Figure 1 Examples of GLUT-1 expression in OSCC as determined by immuno-histochemistry. The paraffin sections demonstrate GLUT-1 staining in the biopsy material of the tumors belonging to: (a) the low LI subgroup (
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Figure 2 Co-expression of GLUT-1 and KI-67 at the outer border of an OSSC. Note that the intense pathologic expression of GLUT-1 demarcates the tumor (b) from the adjacent tissues. Immunohistochemistry of the resection specimen of a non-irradiated tumor. Original magnification, 200·.
Association between GLUT-1 expression in the tumor and patient survival During the follow-up period, 4 of 20 patients (20%) in the ‘‘low LI’’ group (LI < median) and 11 of 20 patients (55%) in the ‘‘high LI’’ group (LI P median) had died. The difference in survival between the two groups was statistically significant (p = 0.044). The Kaplan–Meier plot in Figure 4 suggests that higher GLUT-1 LI predicts a lower survival probability in patients undergoing preoperative radiation therapy. When considered as a single continuous variable in the Cox regression model, GLUT-1 was assigned a hazard ratio of 1.028 per one unit increase of LI. The dichotomized GLUT-1 LI was also confirmed as a significant prognostic marker in a proportional hazards regres-
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GLUT-1 LT [%]
The association between GLUT-1 expression and radiation resistance was also significant for the more homogeneous cohort of 29 locally advanced (T3 and T4) tumors (Figure 3). In this subpopulation, the average LI was 42.7% (±18) for the Rad-R0 group, 53.9% (±17) for the Rad-R1 group and 71.8% (±16) for the Rad-R2 group (p = 0.0116). The difference between clinical responders (groups Rad-R0 + RadR1) and incomplete responders (group Rad-R2) was highly significant (p = 0.004). In addition, the Fisher’s exact test confirmed a significant association between GLUT-1 expression in the tumor and radiation resistance (p = 0.032). These results suggest that GLUT-1 expression could be considered as a marker of radioresistance in OSCC, with a high GLUT-1 expression being associated with poor radiation response and vice versa.
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group Rad-R1
group Rad-R2
Figure 3 GLUT-1 expression stratified according to radiation responses in the homogenous group of 29 OSCC patients with T3 and T4 tumors. Resistance to radiation was associated with an increased GLUT-1 labeling index and vice versa (p = 0.023). The three groups were defined on the basis of histological evaluations as: Group Rad-R0 (n = 3): no residual vital tumor; Group Rad-R1 (n = 9): remaining viable tumor cells; Group Rad-R2 (n = 17): bulky residual tumor (clinical and pathological evaluation). The box plots show median % of LI (black bar); the boxes are interquartile (25–75%) ranges, and the whiskers extend to the maximum and minimum values.
sion analysis, controlled for T-stage, N-stage, grading, and administration of chemotherapy (Cisplatin). The estimated hazard ratio between low and high GLUT-1 LI was 5.11 with
GLUT-1 is associated with resistance to radiotherapy and adverse prognosis 1.0
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LI < median (n=20)
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Time [months] Figure 4 Kaplan–Meier survival estimates generated by univariate analysis for high LI (>median) vs. low LI (
a p-value of 0.035. The hazard ratio, estimates along with 95% confidence intervals and p-values from the Wald tests for all variables are given in Table 1. The data indicate that the GLUT-1 LI could be considered a negative predictive marker of prognosis in preoperatively irradiated OSCC, with a high GLUT-1 LI being associated with an unfavorable outcome. Both the impact of GLUT-1 expression on radiation response and the association between GLUT-1 expression and survival suggest a biologically and clinically relevant influence of glucose metabolism on radiation responses in OSCC.
Discussion The role of hypoxia as a potential factor counteracting the efficacy of radiotherapy has been thoroughly investigated in the last decade.19–21 These studies have established a significant correlation between endogenous markers of hypoxia and clinical outcome.22–24 Only recently, Koukourakis and coworkers have reported a strong association between two
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separate ‘‘hypoxia markers’’ (HIF-2A, CA9) and survival in a large cohort of head and neck cancer patients originally recruited in the CHART-randomized trial.25 There are two fundamental drawbacks when outcome effects are directly attributed to the hypoxia-induced radiation failure. First, the so called ‘‘endogenous markers of hypoxia’’, including HIF, CA9 and GLUT-1, do not exclusively correspond to the tumor oxygenation status. Rather, expression in tumor cells of these markers might reflect the activity of other oncogenic pathways independent of hypoxia. In fact, two recent reports comparing oxygen tension in cervical cancers to HIF-1a and GLUT-1 expression provided strong evidence for a regulation of these pathways independently of hypoxia.26–28 Further Chen et al.28 have identified a hypoxia-independent promotive effect of Ras on the GLUT-1 expression triggered via the HIF-1a binding site in the GLUT-1 promoter region. These results are in agreement with the more recent report by Elstrom and coworkers on hypoxia-independent stimulation of glycolysis by Akt transfection.29 In our hands, a hypoxia-independent up-regulation of GLUT-1 expression is also supported by the characteristic locoregional distribution pattern of staining for GLUT-1 seen in 23/39 tumors. The distinct localization of GLUT-1 staining to the tumor border (see Fig. 2) cannot be explained by a gradient of oxygen saturation between the tumor and the immediate proximate tissue but rather suggests hypoxia-independent overexpression of GLUT-1. The second drawback is a methodological one. The ‘‘true’’ efficacy of radiation therapy has not been verified in previous studies, because no complete or even representative histological controls were performed. The contribution of the cellular markers to survival was exclusively attributed to radiation resistance, although several other biological tumor properties (i.e., tumor cell chemosensitivity or tumor invasiveness) could also have contributed to the clinical outcome. In order to go a step further, our study examined two clinical endpoints with regard to the enhanced expression of GLUT-1 in OSCC: (i) radiation response measured by histological evaluation of complete resection specimens as first-hand information and (ii) overall survival as a confirmative parameter. In our investigation, a consistent and significant influence of GLUT-1 could be verified for both endpoints. The GLUT-1 expression level was inversely correlated to radiation response and, most important, it was predictive of the clinical outcome in both univariate and
Table 1 Survival analysis based on GLUT-1 overexpression in the tumor and controlled for other prognostically important variables in OSCCa Variable
Definition of groups (Group 1 vs. Group 2)
95% Confidence interval for the hazard ratio
Hazard ratio (Group 2 relative to Group 1)
p-value (Wald)
T-stage N-stage Grade of differentiation Chemotherapy (Cisplatin)
T1 + T2 vs. T3 + T4 N0 vs. N > 0 G1 + 2 vs. G3 Administered vs. not administered
0.66–32.21 0.34–24.42 0.13–2.69 2.49–77.15
4.61 2.88 0.60 13.85
0.12 0.33 0.50 0.003
1.12–23.39
5.12
0.035
GLUT-1 (LI) a
Cox regression analysis.
802 multivariate survival analyses, with a high GLUT-1 expression being associated with poor survival. Although the association between GLUT-1 expression and radioresistance has not been established at the clinical level in other human solid tumors to date, in vitro experiments and tumor xenograft studies reported by Pedersen and coworkers argue that GLUT-1 expression plays a hypoxia-independent role in the modulation of radiation susceptibility.11 These investigators demonstrated a linkage between GLUT-1 expression and radiation resistance in two cell sublines (CPH-54A and CPH-54B) derived from a single small cell carcinoma of the lung. Apart from tumor metabolism, the protective effects of glucose transporters and glucose consumption are well known in cardiology.30 Several lines of evidence support the concept that glucose-dependent inhibition of the mitochondrial apoptotic pathway is highly relevant to the protection of cardiac myocytes against apoptosis.30 Additional potential protective mechanisms involving glucose include the cellular redox control by NADH supply31 and caspase-2 inhibition by NADPH generation.13 It is intriguing to note, that radiation-induced cell death is, to a substantial extent, mediated by the same pathways,32 providing support for the hypothesis that cellular glucose metabolism directly affects cellular radiosensitivity. Taken together, our comparative analysis of GLUT-1 expression and therapeutic efficiency of radiation, as measured by histology and overall survival, provide first clinical evidence that GLUT-1 may be a useful parameter for identifying patients with radioresistant OSCC, which could not be expected to be cured by radiotherapy alone. Furthermore, our results confirm the value of GLUT-1 expression as a predictive marker for this tumor type. In addition to providing prognostic information, our data suggest that modulation of radiation resistance by inhibition of glucose transport in the tumor may be a novel strategy to improve effectiveness of radiotherapy in OSCC.
Conflict of interest None declared.
Acknowledgements This work was supported in part by a grant of the ‘‘Mainzer Forschungsfo ¨rderungsprogramm des Fachbereichs Medizin’’ to M. Kunkel (MAIFOR: Glukosetransporter) and by the NIH grant PO1-DE12321 to T.L. Whiteside.
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