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Molecular Oncology |
and Hypoxia-inducible Factor-2 in Human Bladder Tumors and Cell Lines1
Molecular Oncology Unit, Imperial Cancer Research Fund, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS [A.J., C.F., J.W.M., R.B., A.L.H., C.B.]; Department of Urology, Churchill Hospital, Oxford, OX3 7LJ [A.J., D.C.], and Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU [A.J., C.F., S.F.], United Kingdom
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ABSTRACT |
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(HIF-1) and with hypoxia-inducible factor-2 (HIF-2) and in RT4 correlated with a marked reduction in HIF-1 inducibility at confluence. Using the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY 294002, we show that this VEGF hypoxia-inducible pathway regulated by HIF-1 is distinct from a PI 3-kinase-dependent pathway, which regulates basal amounts of VEGF, but does not affect inducibility. Both HIF-1 and HIF-2 protein and mRNA were up-regulated in primary human bladder tumors (n = 12) compared with normal bladder specimens (n = 4), with significant intertumor variation. These results suggest that components of the hypoxia response pathway, including HIF-1 and HIF-2, are important cofactors in the regulation of VEGF in bladder cancer and are therapeutic targets in this disease. |
Introduction |
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Previously, we have demonstrated that VEGF is prognostic in superficial bladder cancer where high mRNA levels predict early stage progression and poor relapse-free survival (6) . Elevated urinary VEGF protein levels provide similar prognostic and diagnostic information (7) , with high levels being associated with high relapse rates and stage progression.
Hypoxia up-regulates VEGF expression (8)
, partly through increasing mRNA transcription and partly through increasing mRNA stability mediated through the RNA-binding protein HuR (9)
. Within tumors, VEGF mRNA colocalizes with hypoxic regions (10)
. The VEGF gene and several other genes regulated by hypoxia and involved in oxygen homeostasis are under the control of the transcription factor HIF-1 (11)
. HIF-1 comprises two subunits from the basic helix-loop-helix PAS domain protein family, HIF-1 and HIF-1ß/ARNT (12)
. mRNA levels of HIF-1 are equivalent in normoxia and hypoxia (13)
, but in hypoxia there is inhibition of the O2-dependent degradation of HIF-1 protein, via the ubiquitin-proteasome pathway (14)
, recently shown to be regulated by the von Hippel-Lindau tumor suppressor gene product (15)
. There is an exponential increase in HIF-1 protein concentration, with decreasing oxygen tension, with a half-maximum response between 1.5 and 2.0% O2 and a maximum response at 0.5% O2 (16)
.
Because the HIF-1ß/ARNT subunit is constitutively expressed and involved in other cellular processes, the HIF-1 subunit is thought to be the specific regulator of the hypoxia response (17)
. Loss of HIF-1 in embryonic stem cells significantly reduced solid-tumor growth, correlating with decreased expression of VEGF during hypoxia (18)
. We and others have demonstrated reduced in vivo tumor growth and angiogenesis with functional impairment of the HIF-1/ARNT pathway, suggesting that the HIF-1 pathway is an important component of oncogenic angiogenesis (19
, 20)
Another member of the helix-loop-helix family is endothelial PAS protein-1 (21, 22, 23)
. Endothelial PAS protein-1 shows 48% homology with HIF-1 and responds to activating and inhibiting stimuli very similarly to HIF-1. It is now known not to be endothelial-specific, as once thought, but to be present in a number of cell types, and it is more frequently termed HIF2 (24)
.
Hypoxia is common in all solid tumors (25)
and also is associated with radiation resistance and poor prognosis (26)
. Therefore, the key upstream signal for VEGF regulation via HIF-1 may be differentially regulated in cancer and may synergize with other mechanisms of VEGF up-regulation. Because of our previous work showing high VEGF associated with recurrence and relapse, we investigated mechanisms regulating VEGF in human bladder cancer cell lines. The cell lines chosen display a range of differentiation phenotypes from well (RT4) through moderate (RT112) to poorly differentiated (253J,EJ28). RT4 and RT112 are routinely used as models of superficial bladder cancer (27
, 28)
, and 253J and EJ28 are used as models of invasive bladder cancer (27, 28, 29)
, allowing us to study responses representing progression from superficial to invasive cancer.
Here we demonstrate that hypoxia and degree of confluence are important regulators of VEGF in bladder cancer cell lines, and regulation differs in superficial versus invasive cell lines. This regulation is effected in part through the transcription factors HIF-1 and HIF-2 and in part through a PI 3-kinase-dependent pathway. We also demonstrate for the first time that both HIF-1 and HIF-2 are differentially expressed in human primary bladder tumors and related to VEGF mRNA expression. Having demonstrated previously the importance of VEGF in bladder tumors, this evidence suggests that the hypoxia response pathway is one in vivo mechanism regulating VEGF and may be a target for therapies.
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Materials and Methods |
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Measurement of VEGF Protein Levels in Cell Supernatant.
Cells were plated in 1.5 ml of media/well in six-well culture plates (Falcon; Becton Dickinson Labware, Oxford, United Kingdom) at a density of between 1 x 105 and 6 x 105, depending on cell type and degree of confluence desired for the start of that experiment. At that stage, culture medium was aspirated and replaced with fresh assay medium. After 16 h of hypoxia (0.1%) or normoxia, medium was collected, centrifuged to remove cellular debris, and stored at -70°C until assayed for VEGF. VEGF assay was performed using a commercially available ELISA kit (R & D Systems, Abingdon Oxon, United Kingdom). Results between wells were standardized according to the cell number/well as measured on an automated Coulter counter (Beckman-Coulter, High Wycombe, Bucks, United Kingdom) and expressed as pg of VEGF protein/ml supernatant/105 cells.
RNA Analysis.
Total RNA was extracted from 
200 mg tissue using TRI reagent (Sigma Chemical Co., Aldrich, United Kingdom).
RNase protection assays, using the method essentially as described by Petersen et al. (31)
, were carried out in duplicate on 10–30 µg of total RNA. [32P]CTP-labeled riboprobes for VEGF (6)
, HIF-1 (accession no. X95580) and HIF-2 (accession no. U81984) were prepared to the highest specific activity possible. To attenuate the signal strength of the highly abundant loading control, U6 small nuclear RNA (accession no. X01366), a riboprobe of significantly lower specific activity, was prepared (32)
.
The protected fragment size for VEGF121 was 517 nt and for VEGF165 and VEGF189 was 439 nt, thus giving two bands on autoradiographs. Those for HIF-1, HIF-2, and the loading control were 206, 210, and 106 nt, respectively. On each gel a positive and negative (tRNA only) control and undigested riboprobes were run. Intensity of signal, quantified on a phosphorimager (Molecular Dynamics, Sunnyvale, CA), was calculated as the ratio of signal of interest:U6 signal to account for minor loading differences.
Protein Extraction and Immunoblot Analysis.
For whole-cell extracts, cells were washed with ice-cold phosphate-buffered saline and collected by scraping. Cell pellets, or tumor samples, were homogenized in extraction buffer [8 M urea, 10% glycerol, 10 mM Tris-HCl (pH 6.8), 1% SDS, 5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 10 µg/ml pepstatin, and 10 µg/ml leupeptin) using an IKA Ultra-Turrax T8 homogenizer (Janke & Kunkel, Staufen, Germany) for 15 s at full speed. Extracts were quantified using the Pierce BCA protein assay (Pierce, Rockford, IL).
For immunoblotting, proteins were separated in SDS 6% polyacrylamide gels and transferred with a semidry blotter Imm-2 (W.E.P. Company, Concord, CA) to Immobilon P membrane (Millipore, Bedford, MA) overnight in 25 mM Tris base, 190 mM glycine, and 15% methanol. Membranes were blocked with 5% fat-free milk, 0.05% Tween 20, in phosphate-buffered saline. For HIF-1 detection, MoAb 28b (21)
was used at 4 µg/ml, and for HIF-2, MoAb 190b (21)
was used at 2 µg/ml. Detection was performed with horseradish peroxidase-conjugated goat antimouse Immunoglobulins (Dako, High Wycombe, United Kingdom) at 1:1000 and ECL (Amersham Corp., Bucks, United Kingdom). After exposure, membranes were stained with Ponceau S to verify equal protein loading and transfer. Analysis of protein expression was performed by densitometry (Flurchemp; Alpha Innotech Corp., San Leandro, CA).
Tumor Samples.
Superficial transitional cell bladder cancer was obtained from patients undergoing transurethral resection of bladder tumor, and invasive transitional cell bladder cancer and normal bladder was obtained from patients undergoing cystectomy at the Churchill Hospital. Immediately after resection, all samples were snap-frozen in liquid nitrogen. After patient histology confirmed the diagnosis, two 200- to 400-mg portions were removed from the stored sample on ice and used for mRNA and protein preparation.
Statistics.
Statistical analysis was performed using Excel (Microsoft). Comparisons between groups were made using Student’s unpaired t test. The level of significance was P < 0.05.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsREFERENCES |
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When the effects of confluence and hypoxia were examined together, confluence abolished hypoxia-induced VEGF up-regulation in the superficial cell line RT4, but did not affect hypoxic induction in the invasive cell lines (253J, P = <0.01; EJ28, P = <0.05). Furthermore, confluence induced hypoxic up-regulation of VEGF in RT112 not seen during exponential growth (P = <0.05). Thus, whereas exponentially the most superficial cell line expressed the most VEGF, at confluence, the invasive cell lines, by virtue of greater hypoxic induction, expressed more. Thus, in three of four cell lines, confluence reduced basal VEGF, but only in the superficial cell line RT4 did confluence abolish hypoxic inducibility.
This suppression of VEGF induction at confluence in the superficial cell line RT4, but not the invasive cell line EJ28, was also seen when cultures were treated with either CoCl2 (100 µM, final concentration) or DFO (100 µM, final concentration), chemical mimics of hypoxia that induce this pathway (Fig. 2)
.
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. To elucidate further the pathways regulating mRNA levels, we analyzed expression of HIF-1 and HIF-2 in normoxia and hypoxia and under different confluence conditions.
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and HIF-2 Expression under Hypoxia and at Confluence. (Fig. 4A)
. HIF-2 was more variable, with very low expression in RT112 (Fig. 4B)
. Within cell lines, there were certain specific differences. In RT4, there was an increase in HIF-1 and HIF-2 with confluence in normoxia, which was not reflected in increased basal VEGF expression. There was strong hypoxic up-regulation of HIF-1 and HIF-2 during exponential growth in RT4. There was much less hypoxic induction at confluence (The fold inducibility of HIF-1 and HIF-2 progressing from normoxia to hypoxia at confluence was only 29% and 35%, respectively, that of hypoxic induction seen during exponential growth). This is consistent with the loss of hypoxic up-regulation of VEGF protein at confluence in RT4.
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in EJ28, which was unaffected by confluence, and some basal expression of HIF-2 in 253J. Both 253J and EJ28 showed strong hypoxic up-regulation of both HIF-1 and HIF-2, and the degree of hypoxic induction during both exponential and confluence growth phases was more similar than in RT4 (The fold inducibility of HIF-1 and HIF-2 progressing from normoxia to hypoxia at confluence was 60% and 50% for 253J and 74% and 61% for EJ28, respectively, that of hypoxic induction seen during exponential growth). Thus, at confluence, maintenance of hypoxic up-regulation of HIF-1 and, to some degree, HIF-2, in 253J and EJ28 corresponded with maintained VEGF inducibility; whereas in RT4 the loss of hypoxic VEGF inducibility corresponded with a reduction in HIF-1 and HIF-2 inducibility.
RT112 expressed very low levels of HIF-2, both basally and under hypoxia, but there was strong hypoxic induction of HIF-1, which was equal under exponential or confluent conditions. This HIF-1 up-regulation was not associated with an increase in VEGF expression during exponential growth but was associated with an increase in VEGF expression at confluence. The findings at the protein level for HIF-1 and HIF-2 expression were not seen at the mRNA level, where there was no difference between normoxic and hypoxic levels (data not shown).
Relation of PI 3-kinase to Hypoxic Regulation of VEGF.
Confluence altered VEGF expression, and PI 3-kinase activity can be regulated by confluence and proliferation. Mazure et al. (33)
showed in transient reporter assays that hypoxic regulation of hypoxia response element constructs was mediated by PI 3-kinase. Therefore, we used the PI 3-kinase inhibitor LY 294002 to analyze interactions of hypoxia and confluence with HIF-1 and VEGF.
During exponential growth, LY 294002 caused a reduction in the expression of basal and, to a lesser degree, hypoxia-induced VEGF in all cases (Fig. 5A
, Table 1
). However despite a reduction in the expression of total VEGF, the inducibility produced by hypoxia was maintained or even increased in the presence of LY 294002. Indeed at confluence, whereas reduction of VEGF was still seen with LY 294002 under basal conditions, those cell lines that at confluence previously demonstrated hypoxic inducibility (RT112, 253J, and EJ28) were still able to induce VEGF under hypoxia, and, because of a greater fold induction, were able to return VEGF levels to those comparable with cells under hypoxia without LY 294002 (Fig. 5B
; Table 1
) This demonstrates that the PI 3-kinase pathway regulates basal amounts of VEGF during exponential growth and at confluence but does not reduce hypoxic inducibility. Therefore, we analyzed the relation between PI 3-kinase inhibitor and HIF-1. The PI 3-kinase pathway did not appear to be directly involved in phosphorylation of HIF-1, because similar bands were seen, and the molecular weight of HIF-1 was unaffected by the presence of LY 294002 (Fig. 6
).
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and HIF-2 Expression in Bladder Cancers. or HIF-2 was up-regulated and the mechanism of up-regulation, HIF-1 or HIF-2 protein and mRNA were measured in 4 normal and 12 bladder cancer cases (6 superficial and 6 invasive). There was increased expression of both HIF-1 and HIF-2 protein in tumor specimens compared with normal bladder (Fig. 7)
. HIF-1 protein was expressed more in invasive tumors than in superficial, although there was significant intertumor variation, with the highest expression actually in one of the superficial tumors (S5). HIF-2 protein expression was greatest in superficial tumors, and those tumors that had high HIF-2 expression tended to have high HIF-1 coexpression.
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, nor was there an obvious association between mRNA levels and protein levels of either HIF-1 or HIF-2. This is consistent with the main regulation of these factors being posttranslational oxygen-mediated degradation. Nevertheless, mRNA was much higher in some tumors (S4, S6, I1, and I2) for either HIF-1 and/or HIF-2 (with equal loading), raising the possibility of oncogene-induced overexpression (34)
. A significant correlation existed between VEGF mRNA and HIF-1 mRNA (Spearman rank correlation, r = 0.68; P = 0.02), raising the possibility of an oncogene or stress response up-regulating both VEGF and HIF-1 mRNA.
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Contrary to other studies that have shown increased VEGF with increasing cell density in human glioblastoma (U87), fibrosarcoma, (HT1080), and colon carcinoma cell lines (36 , 37) , we demonstrated decreased VEGF protein expression in three of four bladder cancer cell lines at confluence under both normoxic and hypoxic conditions when compared with exponential growth. Cells enter a quiescent phase at confluence, with changes in the expression of various cell-surface receptors, transcription factors, and growth factors (38) . Basic fibroblast growth factor, another key angiogenic factor, was also reduced at confluence in renal cancer cell lines (39) , although the mechanism is unknown.
Confluence may produce stresses, such as low glucose and acidosis, that contribute to VEGF regulation. Because the toxicity of hypoxia is proportional to cell density, with nutritional deprivation and acidosis contributing to hypoxia-triggered apoptosis (40)
, the combination of hypoxia and confluence was also examined. A loss of hypoxic inducibility of VEGF at confluence in the superficial cell line RT4, but not in the invasive cell lines 253J and EJ28, and indeed the gain in RT112, suggests that these latter cells may have a malignant growth advantage. Most superficial bladder tumors have a typical papillary structure, increasing the tumor surface area, and this may result in less tumor cell crowding with the preservation of high VEGF expression in these tumors, as we have demonstrated previously (6)
. Invasive tumors are solid, with associated increased cell density and intratumoral hypoxia, but adaptive mechanisms we have described in vitro may ensure that VEGF induction is maintained. Our results analyzing protein expression of HIF-1 and HIF-2 can explain some, but not all, of these findings.
Increased HIF-1 protein generally correlated with increased VEGF expression. One exception to this was RT4, where hypoxic up-regulation of VEGF was lost at confluence. Here, however, the fold induction of HIF-1 going from normoxia to hypoxia was much less at confluence than during exponential growth (Table 1)
. For 253J and EJ28, where this fold hypoxic induction of HIF-1 was maintained exponentially and at confluence, VEGF inducibility was also maintained. The results for RT112 are harder to explain, because an equal fold induction of HIF-1 under hypoxia is not translated to increased VEGF expression exponentially but occurs only at confluence. These results suggest that hypoxic and density-dependent regulation of VEGF is a balance between HIF-1 up-regulation interacting with other factors regulated at confluence (41)
. In addition, RT112, the only cell line to show hypoxic induction at confluence but not exponentially, was also the only cell line without HIF-2. Whether HIF-2 has a specific role in induction under exponential growth will require additional investigation.
The increase in HIF-1 and HIF-2 at confluence in normoxia, seen most strikingly in RT4, but also in 253J and EJ28, may be attributable to mild hypoxia from cell-to-cell contact. Normoxic expression of HIF-1 has only rarely been reported previously (42)
. Integrins mediating cell-to-cell contact also use the PI 3-kinase/AKT pathway via integrin-linked kinase (43)
. Because of these observations and ours that confluence at normoxia (as measured by gas input) up-regulates HIF-1 and HIF-2 but does not increase VEGF, signaling pathways modified by confluence were analyzed. PI 3-kinase activates protein kinase B/AKT and mediates signaling for various growth factors, cytokines, and cell adhesion molecules (44)
. The suggestion that at least two pathways may interact to regulate VEGF expression under hypoxia and confluence is supported by our results with the inhibitor of the PI 3-kinase signaling pathway, LY 294002. This reduced total VEGF expression but still allowed hypoxia inducibility. HIF-1 protein induction was also unaffected by PI 3-kinase inhibition, implying that hypoxia inducibility of VEGF is conferred via HIF-1 but is additive to a basal pathway regulated substantially by PI 3-kinase. This result differs from results of Mazure et al. (33)
, who, using transient transfection of hypoxia response elements into Ha-Ras-transfected NIH3T3R cells and Rat-1 Ras transformed cells, demonstrated significant reduction of VEGF promotor activity under hypoxia with another inhibitor of PI 3-kinase (Wortmannin). They found no effect on basal levels of VEGF promoter activity. In addition, overexpression of a dominant negative mutant of the p85 regulatory subunit of PI 3-kinase inhibited VEGF promoter induction under hypoxia. They concluded that PI 3-kinase mediated VEGF induction through hypoxia response elements. Our results support the findings of Arbiser et al. (45)
that, whereas increased expression of VEGF occurs through cellular PI 3-kinase activity, hypoxic regulation is independent of this. The results on these bladder cell lines differ from two recent papers showing that PI3 kinase modulation, either by expression of PTEN or PI3 kinase inhibitors, reduced hif-1 expression. Clearly, all papers agree on an effect on VEGF mRNA or protein, but it is possible there will be more differences. Thus, in the paper by Zundel et al. (46)
, hypoxia activated PI3 kinase, but in that by Zhong (47)
, it did not. The contributions of LY294002 are similar between studies, but ours used more severe hypoxia: 0.1% versus 1%.
Nevertheless changes in PI 3-kinase activity that affect basal expression would modify the amount of VEGF inducible by hypoxia and be critical in tumor angiogenesis. The additional fold increase in VEGF at confluence in hypoxia in the presence of LY 294002 suggests that a possible inhibitory pathway regulated by confluence may be acting via the PI 3-kinase pathway.
Analysis of human primary tumors revealed greater HIF-1 and HIF-2 protein expression in tumors compared with normal bladder specimens with one or other factor elevated in all tumors. This is the first demonstration of up-regulation of these pathways in human bladder tumors. However, as in the cell lines, there was significant individual variation. Our in vitro data suggest that although HIF-1 and HIF-2 are key components of hypoxic regulation of VEGF, clearly other transcription factors are also important in controlling angiogenesis. As such, and combined with our clinical data, therapy targeting these elements may be of benefit. Variable interactions with apoptotic pathways, angiogenic factors, and other growth factors may explain why experimentally some studies of antagonism of HIF-1 lead to reduced tumor growth (18)
and others show increased tumor growth (48)
.
The use of hypoxia response elements to drive expression of therapeutic genes in response to hypoxia has been demonstrated both in vitro and within solid tumors in vivo (49) . The accessibility of the bladder to intravesical instillation would facilitate delivery of such agents, and our results justify additional investigation of this approach in bladder cancer.
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ACKNOWLEDGMENTS |
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and HIF-2 antibodies. |
FOOTNOTES |
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1 A. J. is supported by the Osman Hill Research Scholarship from the Royal College of Surgeons of England and the British Urological Foundation. J. W. M., R. B., and A. L. H. are supported by the Imperial Cancer Research Fund. 
2 These authors contributed equally to this paper.
3 To whom requests for reprints should be addressed, at Molecular Oncology Unit, Imperial Cancer Research Fund, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, United Kingdom.
4 The abbreviations used are: VEGF, vascular endothelial growth factor; HIF-1, hypoxia inducible factor-1; HIF-2, hypoxia inducible factor-2; PI 3-kinase phosphatidylinositol 3-kinase; PAS, Per Arnt Sim; DFO, desferrioxamine; ECL, enhanced chemiluminescence; ARNT, arylhydrocarbon nuclear translocator; MoAb, monoclonal anitibody.
Received 7/21/00; revised 2/12/01; accepted 2/28/01.
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); exponential growth phase/hypoxia (0.1% O2; 
) confluent growth phase/normoxia (
); or confluent growth phase/hypoxia (0.1% O2; 
). VEGF protein expression in the supernatant (pg/ml), measured by ELISA and standardized per 1 x 105 cells, is presented as means ± SD; n = 3. Comparison between groups by non-paired t test. Representative experiment shown of experiment repeated more than three times.
