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Tumor Biology |
in Clear Cell Renal Carcinomas1
Departments of Nephrology and Medical Intensive Care [M. S. W., P. M. M., A. S., J. S. J., G. G., U. F., K-U. E.] and Urology [J. R., S. A. L.], Charité, Humboldt University, 13353 Berlin, Germany; German Cancer Research Center, 69120 Heidelberg, Germany [I. B., H-J. G.]; Department of Paediatrics, Section of Medical and Molecular Genetics, University of Birmingham B15 2TT, United Kingdom [N. V. M., E. R. M.]; Wellcome Trust Centre for Human Genetics, Oxford OX3 7BN, United Kingdom [P. H. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the regulatory subunit, requires binding to the von Hippel Lindau (VHL) protein. Because functional inactivation of the VHL tumor suppressor gene occurs in up to 70% of clear cell renal carcinomas, we investigated whether this results in overexpression of HIF-1 and its target genes. Immunoblotting revealed increased expression of HIF-1 in 24 of 32 (75%) clear cell renal carcinomas but only 3 of 8 non-clear cell renal tumors. Somatic mutations of the VHL gene were detected only in clear cell renal carcinomas that overexpressed HIF-1. None of the HIF-1-negative tumors displayed a VHL mutation. The level of HIF-1 mRNA was not different between tumors and adjacent kidney tissue. Immunohistochemistry revealed distinct patterns of nuclear staining for HIF-1, depending on histological type and overall abundance of HIF-1. In those clear cell renal carcinomas that showed increased expression on immunoblots, HIF-1 was expressed in almost all cells. In the remaining clear cell and in non-clear cell tumors, staining was focal; these different patterns thus were compatible with genetic stabilization in contrast to microenvironmental stimulation of HIF-1 as the primary mechanism. The mRNA expression of two known target genes of HIF-1, vascular endothelial growth factor and glucose transporter 1, increased progressively with increasing amounts of HIF-1 in tumor extracts. In addition, glucose transporter 1 protein levels correlated with HIF-1 abundance. In conclusion, the data provide in vivo evidence for a constitutive up-regulation of HIF-1 in the majority of clear cell renal carcinomas, which leads to more widespread accumulation of this transcription factor than hypoxic stimulation. These observations are most likely linked to functional inactivation of the VHL gene product. Increased expression of HIF-1 is associated with alterations in gene expression patterns that are likely to contribute to tumor phenotype and progression. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The basic-helix-loop-helix Per-Arnt-Sim domain transcription factor HIF-13
plays a critical role in oxygen homeostasis. It is composed of two subunits, HIF-1 and HIF-1ß. HIF-1 is the oxygen-regulated component that determines HIF activity (2
, 3)
. Its accumulation occurs during hypoxia because of inhibition of its proteolytic degradation through the ubiquitin proteasome pathway (4
, 5)
. HIF-1 transactivates a large number of genes whose protein products either increase oxygen availability or mediate metabolic adaptation to reduced oxygen tensions. Included among these are erythropoietin, glucose transporters, glycolytic pathway enzymes, VEGF, heme oxygenase, and inducible nitric oxide synthase (2
, 3
, 6)
. Experimental evidence indicates an important impact of HIF-dependent gene expression on tumor growth. Tumors derived from cells lacking HIF-1 or HIF-1ß expression manifested significantly reduced vascularization and, in most studies, reduced growth rates as compared with parental cells (7, 8, 9, 10, 11, 12)
.
Several HIF-responsive genes are known to be up-regulated in human malignancies (1)
. In addition, two studies using immunohistochemistry for HIF-1 have recently shown an accumulation of the protein in several types of common human cancers (13
, 14)
. The strongest staining was usually found at the margin between necrotic and viable tumor tissues and at the invading edges of tumors. These localizations are consistent with the known regulation of HIF-1 through hypoxia. In addition, however, in certain tumors, genetic alterations may also be involved in the activation of HIF and its target genes. Most strikingly, cells derived from RCCs express high levels of hypoxia-inducible genes in cell culture independent of oxygenation (15
, 16)
. Following this observation, recent work has linked an up-regulation of HIF to inactivation of the VHL gene (17
, 18)
. RCCs in patients affected by the VHL syndrome and most sporadic RCCs are associated with loss of function of both alleles of the VHL gene (19)
. Experimental introduction of an intact VHL gene into cells derived from renal carcinomas suppresses HIF under normoxic conditions and restores its hypoxic induction, indicating that VHL is essential for destabilization of HIF- subunits (17
, 18)
. Subsequent studies have confirmed that VHL acts as part of an E3 ubiquitin ligase and mediates proteasomal degradation of HIF-1 (20, 21, 22, 23)
. The hypothesis derived from these in vitro findings is that stabilization of HIF-1 may occur independently of regional hypoxia in the majority of clear cell RCCs.
The two recently published series of different human tumors screened for HIF-1 expression included single cases of primary and metastatic RCC (13
, 14
, 18)
. The frequency of nuclear staining for HIF-1 was variable. In one of the surveys staining was noted to be higher and more homogeneous than in other tumors (14)
, which would be consistent with a genetic rather than a microenvironmental stimulation. If functionally relevant, up-regulation of HIF-1 should also be associated with enhanced expression of hypoxia-inducible target genes. Proof of this association might not only be relevant for understanding the progression of RCCs, which are typically highly vascularized and aggressive, but could also exemplify the significance of HIF-dependent gene expression in cancer in general.
We report here an analysis of a series of 40 human renal tumors of different histological type, which provides data strongly supporting the concept of genetic stabilization of HIF and its functional relevance. High-level expression of HIF-1 was detected in the majority of clear cell RCCs and was found to be homogeneous on tissue sections. In contrast, in non-clear cell RCCs, HIF-1 was expressed less frequently, at lower levels, and focally. Furthermore, the expression of HIF-1 was strongly correlated with the induction of two genes known to be regulated by HIF-1 and important for tumor metabolism and angiogenesis: VEGF and GLUT1.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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protein. All tissues were stored at -70°C until analysis. The collection of samples was approved by local ethics committees, and informed consent was obtained from each patient. Renal tumors were routinely processed and classified for their histological type and grade according to Thönes et al. (24)
in institutes of histopathology at Berlin and Heidelberg without knowledge of the study’s intent or results.
Cell Culture.
Protein extracts from the human hepatoma cell line Hep3B were used as a standard for hypoxic induction on each immunoblot for HIF-1. Cells were grown to confluency and then exposed to 4 h of either normoxia or hypoxia (1% O2, 5% CO2, 94% N2). Hypoxic experiments were performed in a NuAire incubator (Zapf, Sarstedt, Germany).
Immunoblotting.
Protein extraction and blotting were performed essentially as described previously (25)
. Sections of the frozen tissue were fractionated, weighed, and homogenized into 20-fold excess of extraction buffer [7 M urea, 10% glycerol, 10 mM Tris-HCl (pH 6.8), 1% SDS, 5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mg/l each of aprotinin, pepstatin, and leupeptin (all chemicals from Sigma Chemical Co.-Aldrich, Deisenhofen, Germany)] with an electric homogenizer (Ultra-Turrax; IKA, Staufen, Germany). Extracts were quantified with the Bio-Rad DC protein assay (Bio-Rad, Munich, Germany), and 60 µg of each extract were resolved on polyacrylamide gels. Proteins were transferred onto Immobilon P membrane (Millipore, Bedford, MA) overnight and probed with anti-HIF-1 monoclonal antibody (Transduction Laboratories, Lexington, KY) or with an anti-GLUT1 affinity-purified polyclonal antiserum (Alpha Diagnostics, San Antonio, TX) at concentrations of 1 µg/ml each. Signals were detected with horseradish peroxidase-conjugated antibodies (DAKO, Ely, United Kingdom) and enhanced chemiluminescence (SuperSignal West Dura Extended; Pierce, Rockford, IL). After analysis, membranes were stained with Coomassie Blue (Sigma Chemical Co.) to verify equal protein loading and transfer. Each blot contained 40 µg of extract from normoxic and hypoxic Hep3B cells for cross comparison. The abundance of HIF-1 was rated semiquantitatively in comparison with nonmalignant tissue and hypoxic Hep3B extract: -, no expression; +, low-level but definite overexpression; ++, intermediate expression; and +++, high-level expression, similar to the abundance of hypoxic Hep3B extract. The amount of GLUT1 in tumor extracts was rated semiquantitatively in comparison with matched nontumorous kidney tissue: -, no overexpression; +, definite overexpression; ++, high-level overexpression.
Immunohistochemistry.
Immunohistochemistry was carried out on 5-µm sections of paraffin-embedded formaldehyde-fixed tissue. HIF-1 was detected by a mouse monoclonal antibody (0.4 µg/ml; Novus Biologicals, Littleton, CO) with the help of the DAKO catalyzed signal amplification system (DAKO, Hamburg, Germany) as described (13)
. Sections were incubated at 95°C for 45 min with target retrieval solution (DAKO). Subsequent steps were performed according to the manufacturer’s instructions.
For single-label immunohistochemistry of GLUT1, the anti-GLUT1 polyclonal rabbit antiserum (Alpha Diagnostics) was used at 100 µg/ml After application of the antibody, the slides were incubated for 2 h at 25°C; a mouse antirabbit antibody was then added at a dilution of 1:50 (M737; DAKO) and incubated at 22°C for 1 h. This was followed by incubation with rabbit antimouse antibody (Z259; DAKO) at a dilution of 1:40, again at 25°C for 1 h. Alkaline phosphatase-conjugated mouse monoclonal antibody diluted 1:40 was then added and incubated at 25°C for 1 h. All dilutions were in PBS (pH 7.6). For staining, slides were exposed for 15 min to a solution of sodium nitrate (28 mM), new fuchsin (21 mM), naphthol-AS-Bl-phosphate (0.5 mM), dimethylformamide (64 mM), and levamisole (5 mM) in 50 mM Tris-HCl buffer (pH 8.4) containing 146 mM NaCl. Sections were counterstained with aqueous hematoxylin before application of gelating mounting medium.
Double-label immunohistochemistry was performed for HIF-1 and keratin as tumor cell marker, or GLUT1. After the presence of HIF-1 was demonstrated as described, sections were washed in water and PBS and then incubated with 10% FCS. Polyclonal rabbit anti-GLUT1 antibody was used at 100 µg/ml and applied to the sections for 18 h at 4°C. Monoclonal mouse antihuman keratin AE1/AE3 (DAKO) was used at a dilution of 1:25. Sections were then washed three times with PBS at 22°C. After subsequent incubations, as detailed for GLUT1 except that for AE1/AE3 antibody, Z259 was omitted, and incubation with alkaline phosphatase mouse monoclonal antibody, sections were developed with Vector Blue R (Camon, Wiesbaden, Germany) to produce a blue color.
Control experiments entailed immunohistology with nonimmune mouse or rabbit IgG, respectively, without primary antibody, or without avidin biotin peroxidase or alkaline phosphatase antibody.
RNA Analysis.
Total RNA was extracted from frozen tissue samples with RNAzol B, according to the manufacturer’s instructions (Biogenesis, Poole, United Kingdom). RNase protection assays were performed essentially as described previously (26)
. 32P-labeled human riboprobes were generated using SP6 or T7 RNA polymerase (Roche Diagnostics, Mannheim, Germany), with following protected fragments: HIF-1, 240 bp (accession no. U224311); VEGF, 140 bp (accession no. M63971); GLUT1, 136 bp (accession no. K03195); and U6 small nuclear RNA, 106 bp (U6sn; accession no. X01366). Radiolabeled riboprobes were protected from RNase digestion by hybridizing to the following amounts of total RNA: HIF-1, 30 µg; VEGF, 20 µg; GLUT1, 30 µg; and U6sn, 1 µg. The latter was used as internal control for each sample, adding 1/10 (i.e., 100 ng) of each reaction to its appropriate sample after RNase digestion. After resolution on polyacrylamide gels, signals were quantified using a phosphor imager (Fujix, BAS 2000; Fuji, Japan). Values were normalized to the control (U6sn). These values were used to calculate the ratio of abundance in tumor to adjacent kidney tissue, shown as fold induction.
VHL Mutation Analysis.
Genomic DNA was extracted with QIAamp purification columns (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Details of the VHL gene mutation analysis techniques have been described previously (27
, 28)
. In brief, PCR was used to amplify genomic DNA for the VHL coding region (covering codons 54–213) and single-strand conformation polymorphism analysis was performed to detect intragenic mutations as described. Fragments producing an aberrant single-strand conformation polymorphism band were sequenced on the ABI 377 semiautomated sequencer. Direct sequencing of the relevant coding region was performed in a similar fashion for those tumors in which a mutation remained unidentified.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protein and mRNA Expression in Homogenates of Renal Tumors and Adjacent Nonmalignant Renal Tissue. was barely detectable in the non-tumor samples by immunoblotting. In contrast, significant overexpression of HIF-1 was found in 27 of 40 (68%) tumor specimens. The frequency and intensity of HIF-1 expression varied with the type of RCC (Fig. 1, A–C)
. Enhanced accumulation of HIF-1 was detected in 24 of 32 (75%) clear cell RCCs but only 3 of 8 (38%) of non-clear cell RCCs. HIF-1 abundance on immunoblots was categorized semiquantitatively by cross-comparison with the signal from the extract of hypoxic Hep3B cells into three groups: low (+), intermediate (++), and high (+++). With this scoring system, HIF-1 expression was intermediate (++) or high (+++) in >60% of clear cell RCCs but only 2 of 8 (25%) of non-clear cell RCCs (Table 1)
. There was no relationship between tumor grading and the abundance of HIF-1 (data not shown).
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is usually degraded within minutes and, on the other hand, renal artery clamping may itself lead to hypoxic induction of HIF-1, we were concerned about the effect of inevitable variations in time until tissue freezing. To address this issue, different tissue blocks of seven tumors, together with blocks of adjacent nonmalignant kidney tissue were kept at room temperature for up to 30 or 60 min before transfer to liquid nitrogen. Six of these tumors were later identified as clear cell RCCs, and one was an oncocytoma. Five of the six clear cell RCCs showed HIF-1 overexpression, and the abundance of HIF-1 in these tumors remained constant during the time period investigated (Fig. 1B)
. In addition, storage ex vivo did not induce HIF-1 in the oncocytoma, the HIF-1-negative clear cell RCC, or any of the nontumorous kidney samples.
To assay for changes in HIF-1 gene expression, total RNA from all specimens was analyzed by RNase protection. HIF-1 mRNA levels were very similar in all samples investigated, with no difference between tumors and nonmalignant kidney tissue, irrespective of histological type or degree of HIF-1 protein expression [Fig. 1D
; mean ratio of normalized signal intensity between tumorous and adjacent nontumorous tissue (± SE), 0.99 ± 0.12 and 1.28 ± 0.31 in clear cell and non-clear cell RCCs, respectively].
Mutation Analysis of the VHL Gene in Clear Cell Renal Carcinoma.
To evaluate whether the HIF status of a tumor correlates with VHL gene inactivation, we performed mutation analysis of the VHL gene for clear cell RCCs for which tumor DNA was available. In total, 27 clear cell tumors could be studied; 21 of these showed HIF-1 overexpression, of which 12 displayed a somatic mutation of the VHL gene (57%). None of the remaining six HIF-1-negative clear cell RCCs showed a mutation of the VHL gene (Table 2)
. The level of HIF-1 expression in the tumors did not seem to correlate with the presence of a VHL mutation.
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. was found in normal kidney tissue. In the different renal tumors examined, two distinct patterns of nuclear staining for HIF-1 were observed (Fig. 2)
. In the subset of clear cell carcinomas that revealed no overexpression of HIF-1 on immunoblots and in non-clear cell carcinomas, immunostaining for HIF-1 was usually focal, with either single cells (Fig. 2A)
or clusters of cells (Fig. 2B)
accumulating the protein in their nuclei. In some of these cases that had areas of intratumoral necrosis, expression was observed in the perinecrotic tissue. Occasionally, as illustrated in Fig. 2B
, immunostaining for HIF-1 was associated with an apparently avascular tumor stroma of a papillary RCC. In sharp contrast, in those clear cell RCCs that showed increased amounts of HIF-1 protein on immunoblots, i.e., the majority of clear cell RCCs, the staining pattern for HIF-1 was rather homogeneous, with virtually all tumor cell nuclei staining positive (Fig. 2, C–F)
. In these tumors, potential physiological stimuli of HIF-1 expression did not seem to influence its expression because labeling was equally strong in perinecrotic and perivascular regions (Fig. 2D)
.
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In clear cell RCCs, comparison of the mRNA abundance in matched samples revealed at least a 2-fold induction in 24 of 32 cases for both genes (Fig. 3A)
. Although the majority of tumors showed overexpression of either both or neither of the two genes, five tumors expressed significantly higher levels of VEGF or GLUT1 mRNA only.
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Relationship between Expression of HIF-1 Protein and Target Genes.
When tumors were grouped according to whether they contained HIF-1 in amounts sufficient to be detected by immunoblotting, a clear association was found with the incidence of increased mRNA expression of the target genes (Table 3)
. Of 24 clear cell RCCs containing HIF-1, 23 overexpressed VEGF and/or GLUT1 mRNA. In contrast, only half of the HIF-1-negative clear cell RCCs showed up-regulation of at least one of these genes. None of the HIF-1-negative non-clear cell RCCs overexpressed either target gene.
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protein in tumor extracts. This relationship is illustrated in Fig. 4
for all tumors investigated. A very similar increase was found when considering the group of clear cell RCC only. Here the fold induction of VEGF and GLUT1 increased from 0.9 (0.5–3.1) and 1.6 (1.2–1.7) in HIF-1-negative to 9.2 (1.9–0.6) and 8.3 (0.1–22.6) in strongly HIF-1-positive cases (median and interquartile range), respectively.
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overexpression, GLUT1 protein was assayed in immunoblots and by immunohistochemistry (Fig. 5)
. Overall there was a close correlation between the abundance of GLUT1 protein, its mRNA, and HIF-1 expression (Table 4)
. In many cases, the levels of induction of HIF-1 and GLUT1 protein were strikingly similar (compare Fig. 1, A and C
, and Fig. 5, A and B
). On tissue sections, GLUT1 antibody specifically stained cell membranes of tumor cells, as described previously (29)
. In clear cell RCCs that were HIF-1 negative on immunoblots, the staining pattern was usually focal (Fig. 5C)
, whereas it appeared rather homogeneous in HIF-1-positive tumors (Fig. 5D)
. Costaining for HIF-1 and GLUT1 showed a significant, albeit incomplete, overlap between nuclear staining for HIF-1 and cell membrane staining for GLUT1 (Fig. 5E)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A major insight into the molecular events leading to this type of tumor was drawn from the high incidence in patients with germ line mutations of the VHL gene. Conforming to Knudsen’s two-hit hypothesis, the second allele is inactivated in these tumors. The great majority (up to 70%) of sporadic clear cell RCCs have inactivation of both copies of the VHL gene, through either somatic mutations or hypermethylation (19
, 32)
. In contrast, VHL mutations or functional inactivation are not a feature of other types of renal cancer. Several mechanisms have been suggested by which the VHL protein may act as a tumor suppressor. One recently identified function of pVHL is the targeting of HIF-1 for ubiquitin-mediated proteasomal degradation (17)
. The ß domain of pVHL binds directly to the oxygen-dependent degradation domain of HIF-1, which is critically important for its destabilization in the presence of oxygen (20, 21, 22, 23)
.
Following these in vitro findings, the present study provides evidence that marked overexpression of HIF-1 protein is a frequent and functionally relevant feature of clear cell RCCs. Several of our observations support the concept that this up-regulation of HIF-1 is produced mainly by a genetic alteration rather than stimulation through a hypoxic microenvironment. The first observation is that the incidence and level of HIF-1 overexpression were much higher in clear cell than in non-clear cell RCCs (Fig. 1
and Table 1
). The second observation is that, in the majority of clear cell RCCs, immunohistochemistry revealed nuclear staining for HIF-1 throughout the tumor. In these cases, virtually every tumor cell, independent of its position in relation to vascular structures or tumor geometry, expressed the protein (Fig. 2, C–F)
. In contrast, in clear cell RCCs in which the amount of HIF-1 was too low to be detected by immunoblotting of tissue homogenates (Fig. 2A)
and in non-clear cell RCCs (Fig. 2B)
, HIF-1 staining was inhomogeneous and focal. In these tumors, preferential expression in perinecrotic and poorly vascularized areas resembled the expression in diverse types of non-renal human tumors, as recently reported by Zhong et al. (13)
and Talks et al. (14)
. This focal expression pattern most likely reflects hypoxic stabilization. In addition, other genetic alterations, trophic stimuli, and microenvironmental conditions other than hypoxia may also play a role in enhancing regional expression of HIF-1 (1)
. Some of the tumors in our series showed regional activation of HIF-1 on histological specimens, but no signals on immunoblots. This is most likely attributable to the lower sensitivity for detecting a protein in whole-tissue homogenates than with high-sensitivity amplification immunohistochemistry.
The third observation is that the incidence of HIF-1 overexpression detectable by immunoblotting was 75% and thus consistent with the expected frequency of loss of function of VHL. To further pursue the relationship between overexpression of HIF-1 and alterations of the VHL gene, we performed mutation analysis. In total, 44% of the screened clear cell RCCs (12 of 27) displayed a somatic mutation of the VHL gene, which occurred at a frequency similar to that reported previously in primary RCC tumors (27)
. Mutations were found only in the pool of clear cell RCCs that showed overexpression of HIF-1, and in none of the HIF-1-negative clear cell tumors. The frequency of VHL mutations in RCCs showing up-regulation of HIF-1 was 57% (12 of 21; Table 2
). In 
15% of all clear cell RCCs, DNA hypermethylation alone has been found to be the mechanism of VHL inactivation (19
, 32)
. In the selected pool of HIF-overexpressing tumors, this frequency can be expected to be higher. A proportion of tumors in our study with HIF-1 overexpression but no identified somatic mutation of the VHL gene should be accounted for by inactivation by promoter hypermethylation (28)
. In addition, VHL-independent mechanisms for HIF dysregulation might have occurred in some tumors. To date, mutations of HIF-1 itself have not been detected in RCCs (33)
. In summary, the mutation analysis showed a good association between VHL inactivation of a tumor and its HIF status, further supporting the concept of a genetic mechanism for HIF-1 stabilization.
An additional observation compatible with genetically determined inhibition of HIF-1 degradation is its ex vivo stability for up to 60 min in five series of samples from clear cell RCCs (Fig. 1B)
. However, the interpretation of this finding is hampered by the lack of a tumor sample expressing HIF-1 initially and degrading rapidly over time, presumably under the influence of functional VHL-mediated degradation. We did not find such a sample in our series of tumors. Nevertheless, together with the finding that changes in the amount of HIF-1 were not related to alterations at the mRNA level (Fig. 1D)
, these observations are compatible with an abnormal stabilization of the HIF-1 protein, which is usually degraded within minutes after reoxygenation in vitro (2
, 3
, 6
, 25)
. On the other hand, although the oxygen tension within these samples can be assumed to be low, these conditions were not sufficient to induce HIF-1 in similar-sized blocks of adjacent nonmalignant kidney tissue or tumors that did not initially overexpress HIF-1. Artificial induction of HIF-1 through renal artery clamping and variable time periods until freezing of tissue, therefore, seems unlikely.
Others have recently reported findings in brain tumors analogous to our observations in RCCs (13
, 34)
. Although HIF-1 was highly expressed in areas adjacent to necrosis in glioblastomas, hemangioblastomas exhibited homogeneous HIF-1 expression throughout highly vascularized and nonnecrotic tumor tissue. Hemangioblastomas are another common manifestation of the VHL syndrome, and loss-of-function mutations in the VHL gene occur in sporadic hemangioblastomas (19
, 35
, 36)
. Taken together, these findings indicate a functional significance of VHL for destabilization of HIF-1 in diverse tumors in vivo.
An additional important finding in our study is the close correlation between HIF-1 protein levels and the expression of two target genes with entirely different physiological functions (Figs. 4
and 5
; Tables 3
and 4
). VEGF plays a central role in tumor neoangiogenesis (37, 38, 39)
. Several previous studies demonstrated overexpression of VEGF in RCCs at the protein and mRNA levels and correlations with microvessel density (e.g., see Refs. 40, 41, 42, 43, 44
). The glucose transporter GLUT1 mediates cellular glucose uptake and thus facilitates anaerobic glycolysis, which is a prerequisite for clonal expansion of cancer cells (1)
. Immunostaining for GLUT1 was demonstrated previously at the plasma membrane of tumor cells in 85% of clear cell RCCs, but not in association with other renal cancer cell types (29)
.
Our findings confirm these observations and strongly suggest that the heterogeneity in the expression pattern of both VEGF and GLUT1 between and within tumors is at least in part governed by the expression of HIF-1. HIF-1 transactivates the promoters of both genes via binding to hypoxia-responsive elements (45
, 46) and has been shown to regulate GLUT1 and VEGF expression in both cultured cells and experimental tumors (2
, 3
, 6)
. The progressive increases of VEGF and GLUT1 mRNA with increasing abundance of HIF-1 protein, as observed in this study (Fig. 4)
, indicate that this regulative pathway has a dominant influence within the diversity of potential stimuli in vivo. Interestingly, the incidence of overexpression and amplitude of induction for VEGF and GLUT1 mRNA were very similar (Fig. 3B
and Table 3
). Of note, however, is that single tumors in our survey were also observed in which only one of the two genes was overexpressed, pointing to additional regulatory mechanisms.
In the present study, we focused on HIF-1 as the regulatory component of HIF-1. HIF-2 is a structurally related alternative dimerization partner of HIF-ß, which can also transactivate reporter genes via HIF DNA recognition sites (6)
. In vitro it is regulated in an oxygen-dependent fashion very similar to that for HIF-1 (25)
. In solid tumors, however, different but overlapping subsets of tumor cells stained for HIF-1 and -2 (14)
. In vitro studies showed that VHL deficiency also stabilizes HIF-2 (17)
. Moreover, overexpression of HIF-2 mRNA has been reported in hemangioblastomas (47)
. It will be interesting, therefore, to address in future studies the extent to which HIF-2 is induced in RCCs, which may contribute to and amplify the induction of HIF-responsive genes.
The activation of those genes investigated in the present study and other HIF-1-dependent genes as a consequence of overexpression of HIF-1 may have a significant impact on tumor progression. In support of this concept, expression of HIF-1 in cervical carcinomas was recently found to be associated with adverse prognosis (48)
. In RCCs, VEGF expression is associated with poor survival (40)
, and VEGF receptor inhibition reduces the growth of human tumors, including RCCs in experimental models (49)
. Suppression of GLUT1 expression in tumor cells also inhibits growth in a nude mouse model (50)
. Nevertheless, therapeutic attempts to inhibit tumor growth by interfering with the function of any single target gene usually are limited by the role of this particular gene within the complexity of tumor biology and hampered by the redundancy of processes such as neoangiogenesis. In this situation, HIF-1, which has several downstream targets that potentially promote tumor growth, is an attractive candidate for therapeutic exploitation. We propose on the basis of our findings that such attempts may be particularly worthwhile in patients with clear cell RCCs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a grant from the German Research Foundation (DFG; Grant EC 87-3) and the United Kingdom Cancer Research Campaign (CRC). 
2 To whom requests for reprints should be addressed, at Department of Nephrology and Medical Intensive Care, Charité, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany. Phone: 49/30 4505 53132; Fax: 49/30 4505 53909; E-mail: kai-uwe.eckardt{at}charite.de
3 The abbreviations used are: HIF, hypoxia-inducible factor; VEGF, vascular endothelial growth factor; RCC, renal cell carcinoma; VHL, von Hippel Lindau; GLUT1, glucose transporter 1.
Received 10/27/00. Accepted 5/ 1/01.
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 A. Agouni, C. Sourbier, S. Danilin, S. Rothhut, V. Lindner, D. Jacqmin, J.-J. Helwig, H. Lang, and T. Massfelder Parathyroid hormone-related protein induces cell survival in human renal cell carcinoma through the PI3K Akt pathway: evidence for a critical role for integrin-linked kinase and nuclear factor kappa B Carcinogenesis, September 1, 2007; 28(9): 1893 - 1901. [Abstract] [Full Text] [PDF]
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 G. Bratslavsky, S. Sudarshan, L. Neckers, and W. M. Linehan Pseudohypoxic Pathways in Renal Cell Carcinoma Clin. Cancer Res., August 15, 2007; 13(16): 4667 - 4671. [Abstract] [Full Text] [PDF]
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 J. Tabernero The Role of VEGF and EGFR Inhibition: Implications for Combining Anti-VEGF and Anti-EGFR Agents Mol. Cancer Res., March 1, 2007; 5(3): 203 - 220. [Abstract] [Full Text] [PDF]
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T. Shinojima, M. Oya, A. Takayanagi, R. Mizuno, N. Shimizu, and M. Murai Renal cancer cells lacking hypoxia inducible factor (HIF)-1{alpha} expression maintain vascular endothelial growth factor expression through HIF-2{alpha} Carcinogenesis, March 1, 2007; 28(3): 529 - 536. [Abstract] [Full Text] [PDF]
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M. Yao, Y. Huang, K. Shioi, K. Hattori, T. Murakami, N. Nakaigawa, T. Kishida, Y. Nagashima, and Y. Kubota Expression of Adipose Differentiation-Related Protein: A Predictor of Cancer-Specific Survival in Clear Cell Renal Carcinoma Clin. Cancer Res., January 1, 2007; 13(1): 152 - 160. [Abstract] [Full Text] [PDF]
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 S. Philipp, J. S. Jurgensen, J. Fielitz, W. M. Bernhardt, A. Weidemann, A. Schiche, B. Pilz, R. Dietz, V. Regitz-Zagrosek, K.-U. Eckardt, et al. Stabilization of hypoxia inducible factor rather than modulation of collagen metabolism improves cardiac function after acute myocardial infarction in rats Eur J Heart Fail, June 1, 2006; 8(4): 347 - 354. [Abstract] [Full Text] [PDF]
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 C. Sourbier, V. Lindner, H. Lang, A. Agouni, E. Schordan, S. Danilin, S. Rothhut, D. Jacqmin, J.-J. Helwig, and T. Massfelder The Phosphoinositide 3-Kinase/Akt Pathway: A New Target in Human Renal Cell Carcinoma Therapy. Cancer Res., May 15, 2006; 66(10): 5130 - 5142. [Abstract] [Full Text] [PDF]
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I. Talon, V. Lindner, C. Sourbier, E. Schordan, S. Rothhut, M. Barthelmebs, H. Lang, J.-J. Helwig, and T. Massfelder Antitumor effect of parathyroid hormone-related protein neutralizing antibody in human renal cell carcinoma in vitro and in vivo Carcinogenesis, January 1, 2006; 27(1): 73 - 83. [Abstract] [Full Text] [PDF]
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 J.-Y. Han, S. H. Oh, F. Morgillo, J. N. Myers, E. Kim, W. K. Hong, and H.-Y. Lee Hypoxia-inducible Factor 1{alpha} and Antiangiogenic Activity of Farnesyltransferase Inhibitor SCH66336 in Human Aerodigestive Tract Cancer J Natl Cancer Inst, September 7, 2005; 97(17): 1272 - 1286. [Abstract] [Full Text] [PDF]
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D. Zagzag, B. Krishnamachary, H. Yee, H. Okuyama, L. Chiriboga, M. A. Ali, J. Melamed, and G. L. Semenza Stromal Cell-Derived Factor-1{alpha} and CXCR4 Expression in Hemangioblastoma and Clear Cell-Renal Cell Carcinoma: von Hippel-Lindau Loss-of-Function Induces Expression of a Ligand and Its Receptor Cancer Res., July 15, 2005; 65(14): 6178 - 6188. [Abstract] [Full Text] [PDF]
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W. M. Stadler, G. Rosner, E. Small, D. Hollis, B. Rini, S. D. Zaentz, J. Mahoney, and M. J. Ratain Successful Implementation of the Randomized Discontinuation Trial Design: An Application to the Study of the Putative Antiangiogenic Agent Carboxyaminoimidazole in Renal Cell Carcinoma--CALGB 69901 J. Clin. Oncol., June 1, 2005; 23(16): 3726 - 3732. [Abstract] [Full Text] [PDF]
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M. R. Morris, D. Gentle, M. Abdulrahman, E. N. Maina, K. Gupta, R. E. Banks, M. S. Wiesener, T. Kishida, M. Yao, B. Teh, et al. Tumor Suppressor Activity and Epigenetic Inactivation of Hepatocyte Growth Factor Activator Inhibitor Type 2/SPINT2 in Papillary and Clear Cell Renal Cell Carcinoma Cancer Res., June 1, 2005; 65(11): 4598 - 4606. [Abstract] [Full Text] [PDF]
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E. B. Rankin, D. F. Higgins, J. A. Walisser, R. S. Johnson, C. A. Bradfield, and V. H. Haase Inactivation of the Arylhydrocarbon Receptor Nuclear Translocator (Arnt) Suppresses von Hippel-Lindau Disease-Associated Vascular Tumors in Mice Mol. Cell. Biol., April 15, 2005; 25(8): 3163 - 3172. [Abstract] [Full Text] [PDF]
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 V. A.J. Kempf, M. Lebiedziejewski, K. Alitalo, J.-H. Walzlein, U. Ehehalt, J. Fiebig, S. Huber, B. Schutt, C. A. Sander, S. Muller, et al. Activation of Hypoxia-Inducible Factor-1 in Bacillary Angiomatosis: Evidence for a Role of Hypoxia-Inducible Factor-1 in Bacterial Infections Circulation, March 1, 2005; 111(8): 1054 - 1062. [Abstract] [Full Text] [PDF]
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B. I. Rini and E. J. Small Biology and Clinical Development of Vascular Endothelial Growth Factor-Targeted Therapy in Renal Cell Carcinoma J. Clin. Oncol., February 10, 2005; 23(5): 1028 - 1043. [Abstract] [Full Text] [PDF]
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W. Y. Kim and W. G. Kaelin Role of VHL Gene Mutation in Human Cancer J. Clin. Oncol., December 15, 2004; 22(24): 4991 - 5004. [Abstract] [Full Text] [PDF]
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W. K. Rathmell, M. M. Hickey, N. A. Bezman, C. A. Chmielecki, N. C. Carraway, and M. C. Simon In vitro and In vivo Models Analyzing von Hippel-Lindau Disease-Specific Mutations Cancer Res., December 1, 2004; 64(23): 8595 - 8603. [Abstract] [Full Text] [PDF]
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W. G. Kaelin Jr. The Von Hippel-Lindau Tumor Suppressor Gene and Kidney Cancer Clin. Cancer Res., September 15, 2004; 10(18): 6290S - 6295S. [Abstract] [Full Text] [PDF]
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 J. S. Isaacs, Y.-J. Jung, and L. Neckers Aryl Hydrocarbon Nuclear Translocator (ARNT) Promotes Oxygen-independent Stabilization of Hypoxia-inducible Factor-1{alpha} by Modulating an Hsp90-dependent Regulatory Pathway J. Biol. Chem., April 16, 2004; 279(16): 16128 - 16135. [Abstract] [Full Text] [PDF]
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B. I. Rini, S. Halabi, J. Taylor, E. J. Small, and R. L. Schilsky Cancer and Leukemia Group B 90206: A Randomized Phase III Trial of Interferon-{alpha} or Interferon-{alpha} Plus Anti-Vascular Endothelial Growth Factor Antibody (Bevacizumab) in Metastatic Renal Cell Carcinoma Clin. Cancer Res., April 15, 2004; 10(8): 2584 - 2586. [Abstract] [Full Text] [PDF]
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 S. Welsh, R. Williams, L. Kirkpatrick, G. Paine-Murrieta, and G. Powis Antitumor activity and pharmacodynamic properties of PX-478, an inhibitor of hypoxia-inducible factor-1{alpha} Mol. Cancer Ther., March 1, 2004; 3(3): 233 - 244. [Abstract] [Full Text]
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E. Kleymenova, J. I. Everitt, L. Pluta, M. Portis, J. R. Gnarra, and C. L. Walker Susceptibility to vascular neoplasms but no increased susceptibility to renal carcinogenesis in Vhl knockout mice Carcinogenesis, March 1, 2004; 25(3): 309 - 315. [Abstract] [Full Text] [PDF]
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K. Datta, J. Li, R. Bhattacharya, L. Gasparian, E. Wang, and D. Mukhopadhyay Protein Kinase C {zeta} Transactivates Hypoxia-Inducible Factor {alpha} by Promoting Its Association with p300 in Renal Cancer Cancer Res., January 15, 2004; 64(2): 456 - 462. [Abstract] [Full Text] [PDF]
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L. Gunaratnam, M. Morley, A. Franovic, N. de Paulsen, K. Mekhail, D. A. E. Parolin, E. Nakamura, I. A. J. Lorimer, and S. Lee Hypoxia Inducible Factor Activates the Transforming Growth Factor-{alpha}/Epidermal Growth Factor Receptor Growth Stimulatory Pathway in VHL-/- Renal Cell Carcinoma Cells J. Biol. Chem., November 7, 2003; 278(45): 44966 - 44974. [Abstract] [Full Text] [PDF]
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 G. Boner, A. J. Cox, D. J. Kelly, A. Tobar, J. Bernheim, R. G. Langham, M. E. Cooper, and R. E. Gilbert Does vascular endothelial growth factor (VEGF) play a role in the pathogenesis of minimal change disease? Nephrol. Dial. Transplant., November 1, 2003; 18(11): 2293 - 2299. [Abstract] [Full Text] [PDF]
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 P. Maxwell HIF-1: An Oxygen Response System with Special Relevance to the Kidney J. Am. Soc. Nephrol., November 1, 2003; 14(11): 2712 - 2722. [Full Text] [PDF]
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Y. Azuma, S.-C. Chou, R. A. Lininger, B. J. Murphy, M. A. Varia, and J. A. Raleigh Hypoxia and Differentiation in Squamous Cell Carcinomas of the Uterine Cervix: Pimonidazole and Involucrin Clin. Cancer Res., October 15, 2003; 9(13): 4944 - 4952. [Abstract] [Full Text] [PDF]
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Y.-G. Yoo, S. H. Oh, E. S. Park, H. Cho, N. Lee, H. Park, D. K. Kim, D.-Y. Yu, J. K. Seong, and M.-O. Lee Hepatitis B Virus X Protein Enhances Transcriptional Activity of Hypoxia-inducible Factor-1{alpha} through Activation of Mitogen-activated Protein Kinase Pathway J. Biol. Chem., October 3, 2003; 278(40): 39076 - 39084. [Abstract] [Full Text] [PDF]
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S. T. Palayoor, P. J. Tofilon, and C. N. Coleman Ibuprofen-mediated Reduction of Hypoxia-inducible Factors HIF-1{alpha} and HIF-2{alpha} in Prostate Cancer Cells Clin. Cancer Res., August 1, 2003; 9(8): 3150 - 3157. [Abstract] [Full Text] [PDF]
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M. Y. Liu, L. Poellinger, and C. L. Walker Up-Regulation of Hypoxia-inducible Factor 2{alpha} in Renal Cell Carcinoma Associated with Loss of Tsc-2 Tumor Suppressor Gene Cancer Res., May 15, 2003; 63(10): 2675 - 2680. [Abstract] [Full Text] [PDF]
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Y. Jiang, W. Zhang, K. Kondo, J. M. Klco, T. B. St. Martin, M. R. Dufault, S. L. Madden, W. G. Kaelin Jr, and M. Nacht Gene Expression Profiling in a Renal Cell Carcinoma Cell Line: Dissecting VHL and Hypoxia-Dependent Pathways Mol. Cancer Res., April 1, 2003; 1(6): 453 - 462. [Abstract] [Full Text] [PDF]
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 H. Teoh, M. Zacour, A. D. Wener, L. Gunaratnam, and M. E. Ward Increased myofibrillar protein phosphatase-1 activity impairs rat aortic smooth muscle activation after hypoxia Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1182 - H1189. [Abstract] [Full Text] [PDF]
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S. J. Welsh, R. R. Williams, A. Birmingham, D. J. Newman, D. L. Kirkpatrick, and G. Powis The Thioredoxin Redox Inhibitors 1-Methylpropyl 2-Imidazolyl Disulfide and Pleurotin Inhibit Hypoxia-induced Factor 1{alpha} and Vascular Endothelial Growth Factor Formation Mol. Cancer Ther., March 1, 2003; 2(3): 235 - 243. [Abstract] [Full Text] [PDF]
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M. Yao, M. Yoshida, T. Kishida, N. Nakaigawa, M. Baba, K. Kobayashi, T. Miura, M. Moriyama, Y. Nagashima, Y. Nakatani, et al. VHL Tumor Suppressor Gene Alterations Associated With Good Prognosis in Sporadic Clear-Cell Renal Carcinoma J Natl Cancer Inst, October 16, 2002; 94(20): 1569 - 1575. [Abstract] [Full Text] [PDF]
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J. S. Isaacs, Y.-J. Jung, E. G. Mimnaugh, A. Martinez, F. Cuttitta, and L. M. Neckers Hsp90 Regulates a von Hippel Lindau-independent Hypoxia-inducible Factor-1alpha -degradative Pathway J. Biol. Chem., August 9, 2002; 277(33): 29936 - 29944. [Abstract] [Full Text] [PDF]
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M. Zatyka, N. F. da Silva, S. C. Clifford, M. R. Morris, M. S. Wiesener, K.-U. Eckardt, R. S. Houlston, F. M. Richards, F. Latif, and E. R. Maher Identification of Cyclin D1 and Other Novel Targets for the von Hippel-Lindau Tumor Suppressor Gene by Expression Array Analysis and Investigation of Cyclin D1 Genotype as a Modifier in von Hippel-Lindau Disease Cancer Res., July 1, 2002; 62(13): 3803 - 3811. [Abstract] [Full Text] [PDF]
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 M. S. Wiesener, M. Seyfarth, C. Warnecke, J. S. Jurgensen, C. Rosenberger, N. V. Morgan, E. R. Maher, U. Frei, and K.-U. Eckardt Paraneoplastic erythrocytosis associated with an inactivating point mutation of the von Hippel-Lindau gene in a renal cell carcinoma Blood, May 15, 2002; 99(10): 3562 - 3565. [Abstract] [Full Text] [PDF]
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K. J. Turner, J. W. Moore, A. Jones, C. F. Taylor, D. Cuthbert-Heavens, C. Han, R. D. Leek, K. C. Gatter, P. H. Maxwell, P. J. Ratcliffe, et al. Expression of Hypoxia-inducible Factors in Human Renal Cancer: Relationship to Angiogenesis and to the von Hippel-Lindau Gene Mutation Cancer Res., May 1, 2002; 62(10): 2957 - 2961. [Abstract] [Full Text] [PDF]
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M. S. Wiesener and K.-U. Eckardt Erythropoietin, tumours and the von Hippel-Lindau gene: towards identification of mechanisms and dysfunction of oxygen sensing Nephrol. Dial. Transplant., March 1, 2002; 17(3): 356 - 359. [Full Text] [PDF]
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R. D. Leek, K. L. Talks, F. Pezzella, H. Turley, L. Campo, N. S. Brown, R. Bicknell, M. Taylor, K. C. Gatter, and A. L. Harris Relation of Hypoxia-inducible Factor-2{alpha} (HIF-2{alpha}) Expression in Tumor-infiltrative Macrophages to Tumor Angiogenesis and the Oxidative Thymidine Phosphorylase Pathway in Human Breast Cancer Cancer Res., March 1, 2002; 62(5): 1326 - 1329. [Abstract] [Full Text] [PDF]
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V. Vukovic, H. K. Haugland, T. Nicklee, A. J. Morrison, and D. W. Hedley Hypoxia-inducible Factor-1{alpha} Is an Intrinsic Marker for Hypoxia in Cervical Cancer Xenografts Cancer Res., October 1, 2001; 61(20): 7394 - 7398. [Abstract] [Full Text] [PDF]
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; CCRC) and non-clear cell RCCs (
; other). Columns indicate median values; bars, interquartile ranges.
