Presence of HIF-1 and related genes in normal mucosa , adenomas and carcinomas of the colorectum Expression of the transcription factor hypoxia-inducible factor 1 -LRB- HIF-1 -RRB- , which plays a key role in cellular adaptation to hypoxia , was investigated in normal colorectal mucosa -LRB- ten -RRB- , adenomas -LRB- 61 -RRB- , and carcinomas -LRB- 23 -RRB- . Tissue samples were analyzed for HIF-1α , its upstream regulators , von Hippel -- Lindau factor , AKT , and mammalian target of rapamycin -LRB- mTOR -RRB- and its downstream targets glucose transporter 1 -LRB- GLUT1 -RRB- , carbonic anhydrase IX , stromal-cell-derived factor 1 -LRB- SDF-1 -RRB- by immunohistochemistry . In normal colorectal mucosa , HIF-1α was observed in almost all nuclei of surface epithelial cells , probably secondary to a gradient of oxygenation , as indicated by pimonidazole staining . The same staining pattern was present in 87 % of adenomas . In carcinomas , HIF-1α was present predominantly around areas of necrosis -LRB- 78 % -RRB- . Active AKT and mTOR , were present in all adenomas , carcinomas , and in normal colorectal mucosa . GLUT1 and SDF-1 were present in the normal surface epithelium of all adenoma cases , whereas in the carcinoma GLUT1 was located around necrotic regions and SDF-1 was present in all epithelial cells . In conclusion , HIF-1α appears to be physiologically expressed in the upper part of the colorectal mucosa . The present observations support that upregulation of HIF-1α and its downstream targets GLUT1 and SDF-1 in colorectal adenomas and carcinomas may be due to hypoxia , in close interaction with an active phosphatidylinositol 3-kinases -- AKT -- mTOR pathway . Introduction Colorectal carcinomas arise from normal epithelial cells of the colorectal mucosa through an adenoma stage . The transition from benign adenomas to malignant carcinomas , occurring in about 5 % of all adenomas , is only beginning to be explored in appreciable detail . Biological changes involved in this process include important signaling pathways like the mammalian target of rapamycin -LRB- mTOR -RRB- -- AKT pathway while recently also a role for hypoxia-driven signaling was shown -LSB- 6 , 14 -RSB- . Both pathways are mediated by the transcription factor hypoxia-inducible factor 1 -LRB- HIF-1 -RRB- as depicted in Fig. 1 . HIF-1 is involved in cellular adaptation to hypoxia in solid tumors , including stimulation of angiogenesis and glycolysis -LSB- 33 -RSB- . HIF-1 consists of two subunits of which the β-subunit is constitutively expressed , whereas HIF-1α is regulated in particular by the oxygen level . Under normoxia , the HIF-1α protein is hydroxylated by prolyl hydroxylases -LSB- 15 , 16 -RSB- , which enables the von Hippel -- Lindau factor -LRB- VHL -RRB- to bind , resulting in ubiquination and rapid degradation by the proteosome -LSB- 27 -RSB- . HIF-1α is overexpressed in many solid tumors -LSB- 38 -RSB- and its presence has been correlated with , i.e. , poor prognosis in breast and colorectal cancer patients -LSB- 4 , 36 -RSB- . Stromal expression of hypoxia-regulated proteins is an adverse prognostic factor in colorectal carcinomas . Fig. 1Schematic representation of the interaction of the analyzed proteins . The PI3K -- AKT -- mTOR pathway is inducing HIF-1α . In addition , the absence of hypoxia prevents HIF-1α from degradation mediated by VHL . When HIF-1α is present , it binds to HIF-1 β in the nucleus . The formed complex activates target genes , like GLUT1 , SDF1 , and CA IX High protein levels of HIF-1α are not only caused by hypoxia but can also be induced by the absence of wild-type protein 53 , mutated VHL and other tumor suppressor genes , as well as by activation of oncogenes like Her2neu -LRB- c-erbB-2 -RRB- -LSB- 24 -RSB- . In addition , growth factors play a role . However , both hypoxia and signaling-mediated HIF-1 stimulation are mediated by the phosphatidylinositol 3-kinase -LRB- PI3K -RRB- -- AKT , and the mTOR pathway -LSB- 8 -RSB- . Interestingly , both the serine -- threonine protein kinase AKT as well as mTOR have been suggested to play a role in an early stage of colorectal carcinogenesis -LSB- 2 , 30 -RSB- . Furthermore , the presence of a non-hypoxic HIF-1 induction has also been suggested , in colorectal adenomas -LSB- 9 -RSB- . Upon HIF-1 activation , multiple target genes become transcriptionally active , including genes involved in anaerobic glycolysis , metabolism , and angiogenesis . This includes glucose transporter 1 -LRB- GLUT1 -RRB- , which facilitates cellular glucose uptake -LSB- 11 , 26 -RSB- and the angiogenesis-associated genes vascular endothelial growth factor and stromal-cell-derived factor 1 -LRB- SDF-1 -RRB- also known as CXCL12 -LSB- 5 -RSB- . Furthermore , carbonic anhydrase IX -LRB- CA IX -RRB- which is prominently induced by hypoxia and is therefore considered to be an intrinsic hypoxia marker , which correlates with a chemical marker of hypoxia , pimonidazole -LSB- 1 , 17 , 23 , 28 -RSB- . An additional function of CA IX is in the regulation of the maintenance of the physiological equilibrium in normal gastrointestinal tissue -LSB- 31 -RSB- . The present study aims to analyze the role HIF-1 in normal colorectal mucosa , adenomas , and carcinomas . To this end , the presence of HIF-1α as well as of its downstream targets GLUT1 , SDF-1 , and CA IX were analyzed in normal colon tissue , colorectal adenomas , and carcinomas . To explore the possible cause of HIF-1 overexpression , i.e. , hypoxia or oncogenic stimulation , both the presence of the chemical hypoxia marker pimonidazole and of the HIF-1α upstream regulators , VHL , AKT , and mTOR , were investigated . Materials and methods Tumor material A total of 84 tissue samples were studied , consisting of 61 colorectal adenomas , 20 of which contained already a focus of carcinoma and are therefore referred to as progressed adenomas , in contrast to the 41 adenomas with dysplasia only , which are referred to as nonprogressed adenomas . In addition , 23 colorectal carcinomas were analyzed . These samples were obtained from 75 patients , 37 of which were female and 38 male . Mean age was 67.8 years -LRB- range 40 -- 89 -RRB- . Normal mucosa samples were obtained from the resection margins of colectomy specimens from ten patients with colorectal cancer . The study was approved by the Institutional Review Board and was in accordance with Dutch medical ethical guidelines . Determination of hypoxic regions Hypoxic regions in mouse tissues were visualized using the Hydroxyprobe-1 kit -LRB- Chemicon International , Temecula , CA , USA -RRB- . These experiments were approved by the animal experimentation ethics committee , according to local and governmental regulations . Briefly , mice were injected intravenously with pimonidazole -LRB- 60-mg/kg body weight -RRB- , which forms adducts from reductively activated pimonidazole in hypoxic cells . Mice were killed 90 min later by asphyxiation in CO2 . The Hydroxyprobe-1 monoclonal antibody was biotinylated using D-biotinyl-ɛ-aminocaproic acid N-hydroxysuccinimide ester -LRB- Boehringer Mannheim -RRB- and was used to stain pimonidazole adducts in formalin-fixed paraffin-embedded tissues according to recommendations by the manufacturer . Immunohistochemistry Immunohistochemistry was performed on 4-μm-thick full-block consecutive sections . Slides were deparaffinized , followed by rehydration , and subsequently staining in batches with the antibodies described in Tables 1 and 2 . HIF-1α staining was performed as described previously -LSB- 3 -RSB- . In brief , antigen retrieval was performed in antigen retrieval solution -LRB- DAKO , Glostrup , Denmark -RRB- for 45 min at 96 °C . The primary antibody against HIF-1α was incubated at a dilution of 1:500 for 30 min at room temperature and the catalyzed signal amplification system -LRB- DAKO -RRB- was used for detecting HIF-1α staining . Table 1Details of the immunohistochemical stainingsAntibodySpeciesCompany and institutesDilutionIncubationAntigen retrievalDetectionHIF-1αMouseBD Pharmingen1/50030 ′ RTWB TRS 97 °C 45 ′ CSAAktRabbitCell Signaling1/50o/n 4 °C WB Citr 97 °C 10 ′ ABCmTORRabbitCell Signaling1/50o/n 4 °C WB Citr 97 °C 10 ′ ABCVHLMouseBD Pharmingen1/10060 ′ RTWB Citr 97 °C 20 ′ PowervisionCA IXMouseLab Harris1/5030 ′ RTnoneEnvision + Glut-1RabbitDAKO1 / 40060 ′ RTWB Citr 97 °C 20 ′ ABCSDF-1MouseR & D systems1/400o/n 4 °C WB Citr 97 °C 10 ′ ABCHydroxyprobe-1MouseChemicon International1/20040 ′ RT0 .01 % pronase in PBS 40 °C for 40 ′ ABCBD Transduction laboratories , Mab monoclonal antibody , PAb polyclonal antibody , RT room temperature , WB water bath , TRS target retrieval solution -LRB- DAKO -RRB- , Citr citrate buffer 10 mM pH 6.0 , CSA catalyzed signal amplification system -LRB- DAKO -RRB- , ABC avidin -- biotinylated peroxidase complex , Powervision from immunologic R&D systemsTable 2Number -LRB- and percentages -RRB- of HIF-1α , P-AKT , P-mTOR , VHL , CA IX , GLUT1 , and SDF-1 expression in nonprogressed and progressed colorectal adenomas and adenocarcinomas in the respective categories of staining intensity Nonprogressed Adenoma , n = 41Progressed adenoma , n = 20Adenocarcinoma , n = 23Kruskal -- Wallis test , P-valueHIF-1Neg6 -LRB- 15 -RRB- 2 -LRB- 10 -RRB- 5 -LRB- 22 -RRB- 0.8 +0 -LRB- 0 -RRB- 2 -LRB- 10 -RRB- 1 -LRB- 4 -RRB- + +7 -LRB- 17 -RRB- 3 -LRB- 15 -RRB- 3 -LRB- 13 -RRB- + + +28 -LRB- 68 -RRB- 13 -LRB- 65 -RRB- 14 -LRB- 61 -RRB- AKTNeg0 -LRB- 0 -RRB- 0 -LRB- 0 -RRB- 0 -LRB- 0 -RRB- 0.1 +12 -LRB- 29 -RRB- 7 -LRB- 35 -RRB- 0 -LRB- 0 -RRB- + +11 -LRB- 27 -RRB- 5 -LRB- 25 -RRB- 10 -LRB- 43 -RRB- + + +18 -LRB- 78 -RRB- 8 -LRB- 40 -RRB- 13 -LRB- 57 -RRB- mTORNeg2 -LRB- 5 -RRB- 0 -LRB- 0 -RRB- a0 -LRB- 0 -RRB- 0.4 +2 -LRB- 5 -RRB- 3 -LRB- 16 -RRB- 0 -LRB- 0 -RRB- + +12 -LRB- 29 -RRB- 6 -LRB- 32 -RRB- 7 -LRB- 30 -RRB- + + +25 -LRB- 61 -RRB- 10 -LRB- 53 -RRB- 16 -LRB- 70 -RRB- VHLNeg0 -LRB- 0 -RRB- a1 -LRB- 5 -RRB- 0 -LRB- 0 -RRB- 0.9 +7 -LRB- 18 -RRB- 0 -LRB- 0 -RRB- 1 -LRB- 4 -RRB- + +12 -LRB- 30 -RRB- 11 -LRB- 55 -RRB- 11 -LRB- 48 -RRB- + + +21 -LRB- 53 -RRB- 8 -LRB- 40 -RRB- 11 -LRB- 48 -RRB- CA IXNeg10 -LRB- 24 -RRB- 4 -LRB- 20 -RRB- 1 -LRB- 4 -RRB- 0.3 +0 -LRB- 0 -RRB- 0 -LRB- 0 -RRB- 0 -LRB- 0 -RRB- + +1 -LRB- 2 -RRB- 2 -LRB- 10 -RRB- 2 -LRB- 9 -RRB- + + +30 -LRB- 73 -RRB- 14 -LRB- 70 -RRB- 20 -LRB- 87 -RRB- GLUT1Neg0 -LRB- 0 -RRB- 1 -LRB- 5 -RRB- 0 -LRB- 0 -RRB- 0.02 +8 -LRB- 20 -RRB- 6 -LRB- 30 -RRB- 0 -LRB- 0 -RRB- + +15 -LRB- 37 -RRB- 4 -LRB- 20 -RRB- 6 -LRB- 26 -RRB- + + +18 -LRB- 44 -RRB- 9 -LRB- 45 -RRB- 17 -LRB- 74 -RRB- SDF1Neg8 -LRB- 20 -RRB- a7 -LRB- 37 -RRB- a0 -LRB- 0 -RRB- < 0.005 +4 -LRB- 10 -RRB- 4 -LRB- 21 -RRB- 0 -LRB- 0 -RRB- + +13 -LRB- 33 -RRB- 2 -LRB- 11 -RRB- 5 -LRB- 22 -RRB- + + +15 -LRB- 38 -RRB- 6 -LRB- 32 -RRB- 18 -LRB- 78 -RRB- aOne case not tested For CA IX staining , no antigen retrieval step was used . Slides were incubated with a mouse primary antibody to CA IX in a 1:50 dilution for 30 min at room temperature . Detection was performed with the Envision + system -- horseradish peroxidase system for mouse primary antibodies -LRB- DAKO -RRB- . GLUT-1 staining was performed with a rabbit polyclonal anti-GLUT1 -LRB- antibody clone A 3536 , DAKO , dilution 1:400 -RRB- without antigen retrieval and subsequently developed with a standard avidin -- biotinylated peroxidase complex -LRB- DAKO -RRB- . Staining procedures for AKT and mTOR were identical . After antigen retrieval , endogenous peroxidase activity was blocked for 10 min in methanol containing 0.3 % hydrogen peroxide . The AKT antibody -LRB- Phospho-Akt -LRB- Ser473 -RRB- -RRB- and mTOR antibody -LRB- Phospho-mTOR -LRB- Ser2448 -RRB- -RRB- both obtained from Cell Signaling -LRB- Danvers , MA , USA -RRB- were incubated overnight at 4 °C in a 1:50 dilution and subsequently detected with a standard avidin -- biotinylated peroxidase complex . SDF-1 staining was performed similar to the AKT and mTOR staining , except for the secondary antibody , which was a goat anti-rabbit antibody in this case . For VHL staining , antigen retrieval was performed and endogenous peroxidase was blocked as the AKT and mTOR staining . Primary antibody against VHL was incubated for 60 min by room temperature , followed by Powervision -LRB- Immunologic , Duiven , the Netherlands -RRB- incubation for 30 min and subsequently the staining was detected with diaminobenzidine -LRB- SIGMA FAST ™ 3,3 ′ - diaminobenzidine tablets , Sigma Aldrich , St. Louis , MO , USA -RRB- . Before the slides were mounted with cover slips , all sections were counterstained for 30 s with Mayer 's hematoxylin and dehydrated in 70 % , 96 % , and subsequently 100 % ethanol and finally in xylene . Evaluation and statistics The intensity of the staining was compared between the normal mucosa , nonprogressed , progressed adenomas , and carcinomas and scored by two investigators . Staining intensity was scored in four classes -LRB- 0 to 3 -RRB- taking the negative control as a reference for score 0 and the positive control -LRB- clear cell renal adenocarcinomas -LRB- Grawitz tumor -RRB- as a reference for score 3 -RRB- . For HIF-1α , only nuclear staining was scored , for CA IX , membrane staining , and AKT and SDF-1 cytoplasmic staining , for mTOR predominantly cytoplasmic but also membrane staining was scored . Significance of differences between categories was analyzed by means of the Kruskal Wallis test . P-values < 0.05 were considered to be significant . Results HIF-1α expression and its regulators hypoxia and VHL In normal colorectal mucosa nine out of ten samples showed HIF-1α present in nuclei of the surface epithelium -LRB- Fig. 2 -RRB- . To evaluate whether hypoxia played a physiologic role in the induction of HIF-1α in normal colorectal mucosa , we used mice that were injected with pimonidazole , a chemical marker of hypoxia that can be visualized by immunohistochemistry . The presence of tissue hypoxia was demonstrated in the surface epithelium of normal colon mucosa of the treated mice -LRB- Fig. 3 -RRB- . The pattern of hypoxic cells in the normal colorectal mucosa in mice matched with the distribution of HIF-1α immunohistochemical staining in human colorectal mucosa . Fig. 2Serial sections of normal colon mucosa , colorectal adenoma , and adenocarcinoma analyzed for HIF-1α and VHL expression . HIF-1α staining was observed in the nuclei of surface epithelium of normal mucosa and colorectal adenoma , whereas in adenocarcinomas HIF-1α was present near hypoxic regions with necrosis . VHL expression was observed in the cytoplasma of all epithelial cells of normal mucosa , adenomas , and adenocarcinomasFig . 3Detection of hypoxia in murine colorectal mucosa by pimonidazole . a Colorectal mucosa of a non-injected mouse , b colorectal mucosa of a mouse that was injected intravenously with pimonidazole . After 90 min , the mouse was killed and colon tissue harvested and formalin fixed . Immunohistochemical staining against pimonidazole revealed hypoxia in the surface epithelium of the normal colon mucosa The presence of HIF-1α in normal colorectal mucosa was compared with that in adenomas and carcinomas . Adenomas displayed HIF-1α expression in 53 of the 61 cases , predominantly at the surface , gradually decreasing towards the center of the adenomas . No differences were seen between nonprogressed and progressed adenomas -LRB- 85 % and 90 % , respectively -RRB- , as shown in Table 2 . Of the carcinomas , 78 % -LRB- n = 18 -RRB- were positive , showing nuclear staining of HIF-1α in epithelial tumor cells . In carcinomas , a decreasing gradient of HIF-1α staining was observed from necrotic areas towards the well-vascularized stroma . Besides the epithelial tumor cells , in carcinomas , inflammatory cells in the tumor-associated stroma , and in five cases B-cells in lymph follicles , were positive for HIF-1α . Statistical analysis showed no significant differences in intensity of HIF-1α staining between normal mucosa , adenomas , and carcinomas . VHL , the regulator of HIF-1α degradation , was also analyzed by means of immunohistochemistry -LRB- Fig. 2 -RRB- . VHL expression was observed in all epithelial cells in normal colorectal mucosa as well as in colorectal adenomas and carcinomas . AKT and mTOR , oncogenic pathways regulating HIF-1α To investigate whether , in addition to hypoxia , oncogenes and/or growth factors can contribute to the elevated HIF-1α , we studied the presence of phosphorylated forms of AKT and mTOR , which reflect an active state of the oncogene-driven pathway of HIF-1α activation . Low-intensity cytoplasmic staining for the active form of AKT -LRB- P-AKT -RRB- was seen in epithelial cells of normal mucosa in all tissues -LRB- Fig. 4 -RRB- . Positive staining was also seen in the smooth muscle cells of the muscularis propria and muscularis mucosae and in the walls of blood vessels . In adenomas , staining in epithelial cells was more intense , present throughout the neoplastic tissue , and observed in all cases . Staining patterns in adenocarcinomas were similar and also observed in all samples analyzed . Fig. 4Typical immunohistochemical staining patterns of the active forms of AKT -LRB- P-AKT -RRB- and mTOR -LRB- P-mTOR -RRB- in normal colon mucosa , colorectal adenoma , and adenocarcinomas . Presence of active AKT is seen in the cytoplasm of all epithelial cells with increasing intensities from the normal mucosa towards adenocarcinoma . Presence of active mTOR is restricted to the surface epithelium in normal mucosa and adenomas . In adenocarcinomas , cells surrounding necrotic areas show active mTOR as well . In addition , in adenocarcinomas , intracellular localization of mTOR changes from cytoplasm towards cellular membrane staining Active mTOR was only present as a cytoplasmatic staining ; nuclear staining was not observed in any of the cases -LRB- Fig. 4 -RRB- . In normal mucosa , mTOR was detected in the surface epithelium in all tissues . Likewise , in adenoma samples , mTOR was expressed in the surface epithelium but also more diffusely throughout the adenomas . In adenocarcinomas , staining patterns were diffused across the whole tumor , but more intense expression was detected in areas surrounding necrosis . Higher levels of active mTOR were observed at the invasive front of the tumor . In the adenocarcinomas , besides the cytoplasmic staining in some cells , a clear cellular membrane staining was observed . In all normal mucosa samples , adenomas , and carcinomas , mTOR was present , except for 5 % of the nonprogressed adenomas , which were negative for mTOR -LRB- Table 2 -RRB- . Expression of the HIF1 downstream targets Glut1 and SDF-1 To investigate to what extent downstream pathways were affected by HIF-1 upregulation , several HIF-1 target genes , including GLUT1 and SDF-1 , were analyzed in normal mucosa , adenomas , and adenocarcinomas . Expression of GLUT1 , which is involved in glucose metabolism , was closely correlated with HIF-1α in all colon tissues analyzed . In normal colon mucosa , GLUT1 expression showed a gradient from the surface epithelium towards the basis of the crypts -LRB- Fig. 5 -RRB- . All normal tissues showed a positive GLUT1 staining . In nonprogressed adenomas , all cases showed GLUT1 expression compared to 95 % of progressed adenomas . All adenocarcinomas -LRB- n = 23 -RRB- were positive for GLUT1 as well . Staining was localized at the cell membrane of epithelial cells in normal mucosa , adenomas , and adenocarcinomas -LRB- Fig. 5 -RRB- . Most intense staining was seen at the surface epithelium of adenomas and in the vicinity of necrosis in carcinomas . As to staining intensity , a significant difference was detected between the adenomas and the carcinomas -LRB- Table 2 -RRB- . Fig. 5Typical immunohistochemical staining patterns of HIF-1α , GLUT1 , SDF-1 , and CA IX in normal colon mucosa , colorectal adenomas , and adenocarcinomas . The downstream targets of HIF-1 , i.e. , SDF-1 , GLUT1 , and CA IX , have predominantly overlapping staining with the presence of HIF-1 . However , in normal mucosa , CA IX is not expressed in the HIF-1α positive surface epithelium but is expressed in the basal of the crypts . In adenocarcinomas , SDF-1 is not only present in HIF-1α-positive cells but in all epithelial cells In normal colon tissue , SDF-1 expression was comparable to that of HIF-1α , i.e. , it was detected in a gradient from the surface epithelium towards the basis of the crypts . However , in all colorectal adenomas and adenocarcinomas , intense SDF-1 expression was observed in the epithelial cells , which was located in the cell membrane and cytoplasm -LRB- Fig. 5 -RRB- . Because this epithelial SDF-1 expression was seen in all epithelial cells , it co-localized only partly with HIF-1α presence . In contrast to the comparable intensity of the HIF-1α staining in adenomas and adenocarcinomas , the SDF-1 staining intensity was much higher in adenocarcinomas than in the adenomas -LRB- Table 2 -RRB- . In addition to the epithelial expression of SDF-1 , endothelial cells of blood vessels stained positive for SDF-1 -LRB- Fig. 6 -RRB- . Fig. 6SDF-1 expression in epithelial and endothelial cells of normal detected by immunohistochemistry Expression of CA IX CA IX was originally identified in gastrointestinal tissues and later recognized as a HIF-1 target gene in many tissues . In normal mucosa , the presence of CA IX actually did not co-localize with that of HIF-1α . CA IX was observed in the bottom parts of the crypts , i.e. , in the proliferative compartment , in contrast to HIF-1α , which was present in the surface epithelium . Seventy percent of the normal tissues showed CA IX expression . In adenomas and particular adenocarcinomas , CA IX and HIF-1 expressions had more resemblance . In adenomas , CA IX was present in a more diffuse pattern throughout the whole adenoma , including the surface epithelium -LRB- Fig. 5 -RRB- . In the adenocarcinomas , CA IX showed a peri-necrotic staining , mainly co-localizing with the HIF-1α expression . Discussion The present study demonstrates the presence of nuclear HIF-1α in the surface epithelium of normal colon and adenomas and in areas associated with peri-necrotic areas of colon adenocarcinoma . The data support a hypoxia-induced HIF-1 cascade , both in normal and neoplastic colorectal mucosa , as suggested by pimonidazole and HIF-1α patterns in association with GLUT1 and SDF-1 in normal colorectal mucosa and adenomas , as well as peri-necrotic HIF-1α and CA IX patterns in colorectal carcinomas . The occurrence of hypoxia around the surface epithelium was confirmed by pimonidazole staining of unaffected murine colon . Furthermore , hypoxia and the oncogenic activation of the AKT -- mTOR pathway both contributed to HIF-1 expression in adenomas and adenocarcinomas . Moreover , the expression patterns of CA IX in normal mucosa and SDF-1 in neoplastic mucosa pointed to additional regulations independent from HIF-1 . The hypoxia -- HIF-1 cascade The presence of HIF-1α under physiological circumstances in the surface epithelium of normal colorectal mucosa observed in the present study may be due to the anaerobic environment in the lumen of the colon , which contributes to a relatively hypoxic state of especially this surface epithelium . Indeed , we observed accumulation of the chemical hypoxia marker pimonidazole particularly in the surface epithelium , indicating low oxygen levels -LSB- 19 -RSB- . The presence of pimonidazole showed a gradient towards the basis of the crypts , suggesting that the cause of hypoxia was indeed related to the anaerobic lumen . Apparently , the capillaries in the surface regions of the colon epithelium were unable to cope with the high oxygen demand of the tissue . This strongly suggests that these hypoxic areas may induce HIF-1α , which contributes to a sustained pressure on the stimulation of angiogenesis and glycolysis to adjust the energy levels of these cells . Although immunohistochemical analysis of patient samples does not allow a formal analysis of functional relationships , it is highly likely that the HIF-1 detected in normal colorectal mucosa was transcriptionally active because HIF-1α was detected in the nuclei and the HIF-1 downstream targets GLUT1 and SDF-1 were present in the same regions where HIF-1α was found . In 87 % of colorectal adenomas , HIF-1α was present , showing staining predominantly in the nuclei of the surface epithelium . In colorectal adenocarcinomas , the presence of HIF-1α changes from the surface epithelium to epithelium cells surrounding regions of necrosis , indicating severe hypoxia . Presence of HIF-1α in the nuclei of epithelial cells went along with expression of the downstream targets GLUT1 and SDF1 in all different stages of the colon carcinogenesis . In contrast to SDF-1 and GLUT1 , the downstream target CA IX , known as a stringent hypoxia marker -LSB- 35 -RSB- , did not co-localize with HIF-1α staining in normal colorectal mucosa . It should be noted that CA IX was originally described to have a role in maintaining the physiological equilibrium in the gastrointestinal tract . In fact , CA IX was more abundant in the basal half of the crypts , containing the proliferating compartment , rather than in the upper half of the mucosa where HIF-1α expression was observed . Indeed , co-localization of CA IX with Ki-67 , a proliferation marker , has been found in colorectal cancer -LSB- 31 -RSB- . The lack of HIF-1α in the CA IX-positive regions indicates an HIF-1α-independent regulation of CA IX in normal colon epithelium . CA IX expression was reported to be also regulated by the transcription factor SP1 -LSB- 18 , 31 -RSB- . Oncogenic upregulation of HIF-1 Oncogene-dependent regulation of HIF-1 can be mediated by the AKT -- mTOR pathway -LSB- 24 , 39 -RSB- . In the present study , in epithelium of all normal colorectal mucosa , a weak staining of P-AKT was observed , confirming data of Itoh et al. -LSB- 14 -RSB- . In all adenomas and adenocarcinomas analyzed , the active -LRB- phosphorylated -RRB- forms of AKT and mTOR were detected as well . Especially active mTOR showed co-localization with HIF-1α in normal tissue as well as in adenomas and adenocarcinomas . Co-localization of P-AKT with HIF-1α was less evident . Recently , in breast cancer , it has been shown that in the absence of P-AKT , only low levels of HIF-1α could be detected while in the presence of P-AKT high HIF-1α protein levels were observed -LSB- 10 -RSB- . In colorectal tumorigenesis , this regulation mechanism may be less relevant because staining was already observed in normal tissue . In addition , the presence of active AKT was not HIF-1 related because in adenocarcinomas P-AKT was present in a higher percentage of cells present than HIF-1α . The downstream target of AKT , mTOR , displayed comparable cellular expression patterns to HIF-1α in normal , adenomas , and carcinomas . This suggests that the AKT -- mTOR -- HIF-1α axis may be important in tumor growth and development as was seen in prostate epithelial cells -LSB- 25 -RSB- . Expression of VHL An alternative mechanism that can lead to HIF-1α overexpression is loss of VHL expression . In normal colon mucosa , VHL is expressed , while in colorectal tumors VHL has been found to be lost or mutated in 11 % of cases -LSB- 7 , 21 -RSB- . Mutations in the VHL gene have been reported to result in cytoplasmic staining of HIF-1α in colorectal cancer in one study -LSB- 22 -RSB- , while this could not be confirmed in another study of 80 patients -LSB- 40 -RSB- . Also in the present study , no cytoplasmic staining of HIF-1 α was seen at all , and VHL expression was seen in all epithelial cells of normal mucosa , colorectal adenomas , and carcinoma . Expression of SDF1 SDF-1 was investigated in the present study as a downstream target of HIF-1 -LSB- 13 , 32 -RSB- . Surprisingly , epithelial cells showed much stronger SDF-1 expression than endothelial cells lining blood vessels . In addition , expression was not merely restricted to areas showing HIF-1α staining but was observed in all epithelial adenomas and adenocarcinomas . SDF-1 staining in tumors cells has also been reported in breast cancer patients -LSB- 29 -RSB- and in clear cell -- renal cell carcinomas , in which HIF-1α is constitutively expressed -LSB- 37 -RSB- . The intense staining of the colon tumor cells suggests that SDF-1 not only has a function in attracting circulating progenitor cells towards the angiogenic areas -LSB- 12 -RSB- . Indeed , SDF-1 stimulates intestinal epithelial cell migration and enhances the integrity of the mucosal barrier -LSB- 34 -RSB- . The level of expression of SDF-1 might also play a role in tumorigenesis because it is related with prognosis in colorectal cancer . The presence of its coding mRNA and the mRNA of its receptor , CXCR4 , have been shown to be a poor prognostic factor -LSB- 20 -RSB- . In tumor formation , SDF-1 may bear impact on the recruitment of various types of progenitor cells needed for enhanced growth of the tumor tissue -LSB- 29 -RSB- . Our study supports a role of SDF-1 in carcinogenesis because SDF-1 staining becomes more intense with the progression of normal colon tissue toward carcinomas . However , future studies are needed to substantiate such role . Conclusion The common expression of HIF-1α in the surface epithelium of the colorectal mucosa suggests that HIF-1 may play a role in the physiology of normal colon tissue . HIF-1α presence does not appear to be related to progression of colorectal carcinogenesis in contrast to GLUT1 and SDF-1 , which are expressed more intensely at later stages of this process .

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