Reversible post-translational modification of serine and threonine residues by
Under normal conditions, 2–3% of glucose taken up by the cells is shunted into the hexosamine biosynthesis pathway, which is a nutrient-sensing pathway that produces uridine diphospho (UDP)-GlcNAc, an energy substrate of OGT, as an end product (
Recent meta-analyses in epidemiological studies have demonstrated that T2DM is a risk factor for colorectal cancer (
Among successive cases histologically diagnosed as colorectal cancer and subjected to surgical resection at our facility between 2008 and 2015, those patients whose condition could be followed up on the basis of clinical information in their medical records were retrospectively analyzed in this study. Patients with a history of treatment for T2DM were classified into the DM group, and patients with no history of treatment for T2DM were classified into the non-DM (NDM) group. Tumor and adjacent non-tumor tissue samples were then examined to determine correlations between the degree of
SW480 human colon cancer cells were obtained from the American Type Culture Collection and maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS containing 100 µg/ml streptomycin and 100 U/ml penicillin. Another human colon cancer cell line, LoVo cells, were obtained from Riken Bioresource Center (Riken BRC). Cells were maintained in DMEM/10% FBS containing 100 µg/ml streptomycin and 100 U/ml penicillin. Both cells were incubated at 37°C in 5% CO2 and 95% air.
Paraffin-fixed tumor and adjacent non-tumor tissue sections cut from surgical blocks were used for immunohistochemistry. The sections were deparaffinized using xylene, dehydrated using ethanol, and activated by microwaving three times in citrate buffer. Next, the sections were blocked using 3% H2O2 solution and M.O.M blocking reagent (Vector Laboratories). Next, the sections were stained using anti-
The immunostained tissue sections were observed under a microscope, and three randomly chosen fields of view were photographed under the same conditions. All imaging analyses were performed using ImageJ software [National Institutes of Health (NIH)]. For evaluation of
Cells were lysed in 1% NP40, 0.25% deoxycholic acid, 0.1 M NaCl, and 25 mM Tris-HCl (pH 7.4). The lysates were subjected to SDS-PAGE and transferred to a PVDF membrane (EMD Millipore) using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories, Inc.). The membrane was probed with antibodies of interest and exposed with Fusion FX (Vilber-Lourmat). The results were densitometrically analyzed with ImageJ software.
Cell lysates were incubated with SureBeads protein G (Bio-Rad Laboratories, Inc.) pre-incubated with anti-β-catenin antibody (#8480, Cell Signaling Technologies, Inc.). The precipitates were eluted according to manufacturer's instructions and subjected to western blot analysis using anti-
SW480 or LoVo cells were seeded in 6-well plates (Thermo Fisher Scientific, Inc.) at 1×105 cells/well in high-glucose (25 mM) growth medium and cultured for 12 h. Next, the cells were cultured in the presence of 1 µM TMG (Cayman Chemical Chemical Company), an OGA inhibitor, for another 36 h. Next, cell lysates were prepared and subjected to western blot analysis.
Three siRNAs against OGA (si
Statistical analyses were performed using GraphPad Prism 6 software (GraphPad Software, Inc.) as indicated in the figure legends (
Thirty-one and 30 patients were enrolled in the DM and NDM group, respectively. No significant differences were found between the two groups in terms of mean age, tumor volume, tumor localization, macroscopic type, cancer stage, and presence or absence of metastasis to other organs. Body mass index was significantly higher in the DM group, and although there were no significant differences in sex between the two groups, there tended to be more men in the DM group (
Next, we examined O-GlcNAc expression in colorectal cancer and adjacent non-cancerous tissues in patients of the DM and NDM groups.
Next, we examined histological scores of O-GlcNAcylation in the DM and NDM groups according to colorectal cancer stage. In the DM group, the histological score increased with cancer progression, revealing a correlation between cancer stage and histological score. In contrast, such a tendency was not found in the NDM group (
In the TNM classification established by the Union for International Cancer Control (UICC), cancer is staged on the basis of tumor size and depth of invasion (T factor), lymph node status (N factor), and presence or absence of distant metastasis (M factor). To examine which of these factors promoted
Using immunohistochemical staining, we examined the expression of β-catenin, which is involved in cancer progression, in patients with colorectal cancer with or without T2DM. In patients with less advanced cancer stages (e.g., stage IIA), no differences were found between the two groups in terms of immunoreactivity; however, in those with advanced cancer stages (e.g., stage IV), staining for β-catenin was stronger in cases of colorectal cancer with comorbid T2DM than in those without T2DM. In addition, β-catenin expression was higher in patients with advanced cancer than in patients with earlier cancer (
Next, we examined whether the β-catenin/SNAIL signaling pathway is upregulated by augmented O-GlcNAcylation and whether the upregulation is amplified in high-glucose condition, using SW480 cells. After the cells were cultured in the presence or absence of 1 µM TMG in normal- or high-glucose condition for 36 h, the expression levels of β-catenin and SNAIL were determined by western blot analyses. While both TMG treatment and high glucose significantly increased
It is well known that patients with T2DM are at significantly higher risk for many types of cancer, but potential biological links between the two diseases are not fully elucidated. Here, we showed one of the mechanisms through which T2DM influences the prognosis of colorectal cancer patients. There was a significant correlation between
Epithelial-mesenchymal transition (EMT) is believed to be required for cancers to undergo metastasis (
Abnormal activation of the β-catenin/SNAIL signaling pathway has been found in various types of malignant tumors, including colorectal cancer (
Our findings suggest that
In conclusion, we found that
The authors would like to thank Ms. Nozomi Tokuhara (Osaka Medical College, Japan) for technical assistance.
This study was supported by the KAKEN grant (grant no. 16K09296) from the Japan Society for the Promotion of Science, and the OMC Internal Research Grant.
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
TO, MA and KH designed the current study. YN, TO, TN, EK, YK, YT, HT, YHira, KKaw, and KKak conducted the current study. TI, TT, SF, YHiro and KU analyzed the data. TO, TN, and MA wrote the manuscript. KH revised the manuscript. All authors have read and approved the final version of the manuscript.
This study was approved by the ethics review committee of Osaka Medical College. All patients provided written informed consent.
Not applicable.
The authors declare that they have no competing interests.
β-catenin expression is enhanced in advanced-stage cancer in patients with T2DM. (A) Immunohistochemical staining for β-catenin. Magnification, ×200. DM, colorectal cancer tissues from patients with T2DM; NDM, colorectal cancer tissues from patients without T2DM. (B) Quantitative analysis of β-catenin expression based on immunohistochemical staining. Four cases from each of the DM and NDM groups for patients with stage IV or stage IIA cancer were analyzed. β-catenin expression was defined as the integrated density of brown color from immunohistochemical staining measured using ImageJ software. *P<0.0001, **P<0.0001, Tukey's test. T2DM, type 2 diabetes mellitus.
TMG treatment in high-glucose condition induces the β-catenin/SNAIL signaling pathway. SW480 human colon cancer cells were cultured in the presence or absence of 1 µM TMG for 36 h in normal (5 mM) or high (25 mM) glucose. (A-C) The cells were lysed and subjected to western blotting with (A) anti-
siRNA-mediated silencing of
Patient characteristics.
Characteristic | DM | NDM | P-value |
---|---|---|---|
Number of cases | 31 | 30 | |
Age, years, mean ± SD | 68.2±10.7 | 70.0±8.2 | 0.460 |
Sex | 0.600 |
||
Female | 7 (22.6%) | 14 (46.7%) | |
Male | 24 (77.4%) | 16 (53.3%) | |
BMI, mean ± SD | 23.9±3.4 | 21.4±3.5 | 0.007 |
Volume, mm2, mean ± SD | 1,497±1080 | 1,740±1279 | 0.43 |
Location | 0.88 |
||
Rectum | 15 (48.4%) | 17 (56.7%) | |
Sigmoid | 10 (32.3%) | 5 (16.6%) | |
Descending | 0 (0%) | 2 (6.7%) | |
Transverse | 3 (9.7%) | 4 (13.3%) | |
Ascending | 2 (6.5%) | 2 (6.7%) | |
Cecum | 1 (3.1%) | 0 (0%) | |
Type | 0.82 |
||
0 | 3 (9.7%) | 3 (10%) | |
1 | 0 (0%) | 0 (0%) | |
2 | 22 (71%) | 24 (80%) | |
3 | 5 (16.1%) | 2 (6.7%) | |
5 | 1 (3.2%) | 1 (3.3%) | |
Stage (UICC) | 0.94 |
||
0 | 0 (0%) | 1 (3.3%) | |
I | 5 (16.1%) | 4 (13.3%) | |
II | 15 (48.4%) | 7 (23.4%) | |
III | 5 (16.1%) | 13 (43.3%) | |
IV | 6 (19.4%) | 5 (16.7%) | |
Metastasis | 1.0 |
||
− | 25 (80.6%) | 25 (83.3%) | |
+ | 6 (19.4%) | 5 (16.7%) |
Student's t test
Fisher's exact test
two-way ANOVA. SD, standard deviation; BMI, body mass index; UICC, Union for International Cancer Control.