Rectal cancer patients and materials
Patients were from the South-East Swedish Health Care region and participated in the randomized Swedish Rectal Cancer Trial of preoperative RT between 1987 and 1990 (Swedish Rectal Cancer Trial, 1997 (3)). Every participant signed the informed consent. The patient cohort of FOXO3 included 143 primary rectal adenocarcinomas, 124 normal mucosa specimens (112 corresponding to the primary cancer, i.e., normal mucosa and primary cancer from the same patient) that were histologically free from cancer and taken from the margin of distant surgical resection, and 50 lymph node metastases (47 corresponding to the primary cancer, also in the irradiation field). Of the 143 patients (median age, 69 years), 79 underwent surgery alone and 64 received RT followed by tumour resection. RT was given with 25 Gy in 5 fractions within a median of 7 days (range, 4–12 days). Surgery was then carried out in a median of 3 days (range, 0–11 days) after RT. None of the patients received preoperative or adjuvant chemotherapy. The mean follow-up period was 105 months (range, 0-309 months), and information on local and distant recurrence; disease free survival and overall cancer-specific survival (OS) were obtained from patient medical records. The patient cohort of FOXM1 included 141 primary rectal adenocarcinomas, 103 normal mucosa specimens (85 corresponding to the primary cancer, i.e., normal mucosa and primary cancer from the same patient) that were histologically free from cancer and taken from the margin of distant surgical resection, and 45 lymph node metastases (41 corresponding to the primary cancer, also in the irradiation field). Of the 141 patients (median age, 69 years), 77 underwent surgery alone and 64 received RT followed by tumour resection. All the rest of information is the same as FOXO3. The patient cohort of SIRT6 included 145 primary rectal adenocarcinomas, 120 normal mucosa specimens (84 corresponding to the primary cancer, i.e., normal mucosa and primary cancer from the same patient) that were histologically free from cancer and taken from the margin of distant surgical resection, and 49 lymph node metastases (45 corresponding to the primary cancer, also in the irradiation field). Of the 145 patients (median age, 69 years), 80 underwent surgery alone and 75 received RT followed by tumour resection. All the rest information is the same as FOXO3. All the characteristics of the patients and tumours of these three factors are presented in Table 4 (FOXO3), Table S3 (FOXM1) and Table S4 (SIRT6).
Tissue samples and immunohistochemistry (IHC)
Expression of FOXO3, FOXM1 and SIRT6 protein was examined by IHC in 4 µm tissue microarray sections from paraffin-embedded surgical specimens. Sample sections were deparaffinized by immersing the slides twice in 100% xylene at room temperature for 10 minutes each. This was followed by incubating twice in 100% ethanol for 10 minutes each, and rehydrating with decreasing concentrations of ethanol (90% and 70%; vol/vol in water, 10 minutes each) before a final 5-minute incubation in water. Antigen retrieval was carried out in a target retrieval citrate buffer (pH 6.0) (Dako, Glostrup, Denmark) at 95 °C for 15 minutes. The sections were allowed to cool for 15 minutes and rinsed with phosphate-buffered saline (PBS) followed by incubation in 3% H2O2-methanol for 5 minutes to block the activity of endogenous peroxidase. After being washed in PBS, the sections were incubated with protein block (Dako) for 10 minutes to reduce nonspecific background staining. The sections were incubated with primary antibody overnight. After being washed in PBS, the sections were incubated with a secondary antibody, Envision System Labelled Polymer-HRP Anti-Rabbit (Dako) for 25 minutes. The sections were rinsed in PBS before reacting with Liquid DAB+ (Dako) to produce coloration. Finally, the sections were lightly counterstained with haematoxylin.
The immunostaining was scored by two independent observers based on the intensity and localization. Staining intensity in normal epithelial cells or tumour cells was graded according to the following criteria: 0 (no staining); 1 (weak staining = light yellow); 2 (moderate = yellow brown); 3 (strong = brown); and 4 (very strong = dark brown). The staining patterns were graded as cytoplasmic or nuclear. In case of discrepancy, a consensus score was reached after re-evaluation. For statistical analyses, cases scored less than 2 were considered as low-expressing group, and cases scored higher than 3 as high-expressing group. The data regarding IHC expression of NF-κB, p53, p73, survivin, Cox2 and PPAR-delta was obtained at our laboratory on the same patient samples as in the present study. The used cut-off points were the same as previous corresponding publications (25–28).
cBioPortal database analysis
The cBio Cancer Genomics Portal (cBioPortal) (http://www.cbioportal.org/) provides visualization tools for more than 5,000 tumour samples from 232 cancer studies in the TCGA database (29). In this study, the Colorectal Adenocarcinoma (TCGA, Firehose Legacy, n = 640) cohort was analysed to explored the genetic alterations of FOXO3, FOXM1 and SIRT6.
MEXPRESS tool analysis
The MEXPRESS tool (https://mexpress.be) is a user-friendly online tool visualizing TCGA data, contains the information on mRNA expression, DNA methylation, clinical data as well as the relationships among these parameters (30). The detail methylation locations of FOXO3 in colorectal cancer were assessed using the MEXPRESS.
GeneMANIA analysis
GeneMANIA (http://www.genemania.org) is a friendly web server for deriving hypotheses based on gene functions (31). GeneMANIA was adopted to conduct a gene-gene interaction network for FOXO3, FOXM1 and SIRT6.
The Cancer Regulome tools and data analysis
The Cancer Regulome tools and data (http://explorer.cancerregulome.org/) from the TCGA dataset were performed to draw circus plots to show the genomic location of FOXO3 and its related-genes in colorectal cancer (n = 621). Spearman correlation was used to show the pairwise correlation between two genes. The circus plots only display the genes with P-values > log10.
Functional enrichment analysis
The Spearman correlation analysis was conducted by the LinkedOmics database (http://www.linkedomics.org/) (32). Spearman’s correlation coefficient exceeding 0.4 indicates a good correlation between FOXO3 and its related genes. Metascape online software (http://metascape.org) was used to construct the interaction network of enrichment terms. All analyses were performed with default software parameters (33).
Statistical analyses
All statistical analyses were performed using STATISTICA software package (version 12.0; STATSOFT Inc., Tulsa, OK). McNemar’s or Person χ2 test was used to examine the significance of the differences in FOXO3/FOXM1/SIRT6 expression among normal mucosa, primary tumour and lymph node metastasis, as well as the association of FOXO3/FOXM1/SIRT6 expression with clinicopathological or biological variables. The survival curves were plotted using Kaplan–Meier analysis and the differences between the curves were calculated by Log rank test. Univariate and multivariate analyses were performed by Cox proportional hazards regression analysis (likelihood ratio test). All tests were two sided and P-values < 0.05 were considered statistically significant.