Introduction 6 14 1 33 15 16 27 38 4 36 Fig. 1 Schematic 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  24 8 2 30 9 11 26 5 1 17 23 28 31 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 (range 40–89). 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 (Chemicon International, Temecula, CA, USA). These experiments were approved by the animal experimentation ethics committee, according to local and governmental regulations. intravenously 2 N Immunohistochemistry 1 2 3 Table 1 Details of the immunohistochemical stainings Antibody Species Company and institutes Dilution Incubation Antigen retrieval Detection HIF-1α Mouse BD Pharmingen 1/500 30′ RT WB TRS 97°C 45′ CSA Akt Rabbit Cell Signaling 1/50 o/n 4°C WB Citr 97°C 10′ ABC mTOR Rabbit Cell Signaling 1/50 o/n 4°C WB Citr 97°C 10′ ABC VHL Mouse BD Pharmingen 1/100 60′ RT WB Citr 97°C 20′ Powervision CA IX Mouse Lab Harris 1/50 30′ RT none Envision+ Glut-1 Rabbit DAKO 1/400 60′ RT WB Citr 97°C 20′ ABC SDF-1 Mouse R&D systems 1/400 o/n 4°C WB Citr 97°C 10′ ABC Hydroxyprobe-1 Mouse Chemicon International 1/200 40′RT 0.01% pronase in PBS 40°C for 40′ ABC BD Mab PAb RT WB TRS Citr CSA ABC Powervision Table 2 Number (and percentages) 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   n n n P HIF-1 Neg 6 (15) 2 (10) 5 (22) 0.8 + 0 (0) 2 (10) 1 (4) ++ 7 (17) 3 (15) 3 (13) +++ 28 (68) 13 (65) 14 (61) AKT Neg 0 (0) 0 (0) 0 (0) 0.1 + 12 (29) 7 (35) 0 (0) ++ 11 (27) 5 (25) 10 (43) +++ 18 (78) 8 (40) 13 (57) mTOR Neg 2 (5) a 0 (0) 0.4 + 2 (5) 3 (16) 0 (0) ++ 12 (29) 6 (32) 7 (30) +++ 25 (61) 10 (53) 16 (70) VHL Neg a 1 (5) 0 (0) 0.9 + 7 (18) 0 (0) 1 (4) ++ 12 (30) 11 (55) 11 (48) +++ 21 (53) 8 (40) 11 (48) CA IX Neg 10 (24) 4 (20) 1 (4) 0.3 + 0 (0) 0 (0) 0 (0) ++ 1 (2) 2 (10) 2 (9) +++ 30 (73) 14 (70) 20 (87) GLUT1 Neg 0 (0) 1 (5) 0 (0) 0.02 + 8 (20) 6 (30) 0 (0) ++ 15 (37) 4 (20) 6 (26) +++ 18 (44) 9 (45) 17 (74) SDF1 Neg a a 0 (0) <0.005 + 4 (10) 4 (21) 0 (0) ++ 13 (33) 2 (11) 5 (22) +++ 15 (38) 6 (32) 18 (78) a 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 (DAKO). GLUT-1 staining was performed with a rabbit polyclonal anti-GLUT1 (antibody clone A 3536, DAKO, dilution 1:400) without antigen retrieval and subsequently developed with a standard avidin–biotinylated peroxidase complex (DAKO). 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 (Phospho-Akt (Ser473)) and mTOR antibody (Phospho-mTOR (Ser2448)) both obtained from Cell Signaling (Danvers, MA, USA) 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 (Immunologic, Duiven, the Netherlands) incubation for 30 min and subsequently the staining was detected with diaminobenzidine (SIGMA FAST™ 3,3′-diaminobenzidine tablets, Sigma Aldrich, St. Louis, MO, USA). 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 P Results HIF-1α expression and its regulators hypoxia and VHL 2 3 Fig. 2 Serial 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 adenocarcinomas  Fig. 3 a b  2 n 2 AKT and mTOR, oncogenic pathways regulating HIF-1α 4 Fig. 4 P AKT P mTOR  4 2 Expression of the HIF1 downstream targets Glut1 and SDF-1 5 n 5 2 Fig. 5 Typical 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  5 2 6 Fig. 6 SDF-1 expression in epithelial and endothelial cells of normal detected by immunohistochemistry  Expression of CA IX 5 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 19 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. 35 31 18 31 Oncogenic upregulation of HIF-1 24 39 14 10 25 Expression of VHL 7 21 22 40 Expression of SDF1 13 32 29 37 12 34 20 29 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.