|Year : 2022 | Volume
| Issue : 3 | Page : 650-655
Expression of glucose transporter 1 (SLC2A1) – Clinicopathological associations and survival in an Indian cohort of colorectal cancer patients
Smreti Vasudevan1, Anurag Mehta2, Sanjeev Kumar Sharma3, Anurag Sharma1
1 Department of Research, Rajiv Gandhi Cancer Institute and Research Centre, New Delhi, India
2 Department of Research; Department of Laboratory and Transfusion Services, Rajiv Gandhi Cancer Institute and Research Centre, New Delhi, India
3 Department of Laboratory and Transfusion Services, Molecular Laboratory, Rajiv Gandhi Cancer Institute and Research Centre, New Delhi, India
|Date of Submission||08-Jan-2021|
|Date of Acceptance||09-Sep-2021|
|Date of Web Publication||27-Apr-2022|
Department of Research, Rajiv Gandhi Cancer Institute and Research Centre, Rohini, Delhi - 110 085
Source of Support: None, Conflict of Interest: None
Background: Glucose transporter 1 (GLUT1) facilitates the uptake of glucose in malignant cells. We investigated GLUT1 transcript expression in colorectal cancer (CRC) tumors and explored its relationship to clinicopathological features, diabetes condition, and patient survival.
Materials and Methods: The expression of GLUT1 was determined using fluorescent probe-based quantitative real-time polymerase chain reaction assay of tumor tissue and corresponding normal mucosa from 180 archived formalin-fixed, paraffin-embedded tissue blocks of ninety upfront surgically resected colorectal adenocarcinoma cases. Clinical information was collected from the hospital medical records and statistical analyses were performed.
Results: Compared to normal mucosa tissue, the GLUT1 expression was significantly elevated in CRC tumor tissue (0.024 ± 0.056 vs. 0.004 ± 0.005; P < 0.0001). The expression was significantly more in poorly differentiated tumors than well/moderately differentiated tumors (P = 0.024) and in patients with liver metastasis (P = 0.013). The high GLUT1 expression correlated with advanced tumor stage (P = 0.003), liver metastasis (P = 0.003), poor tumor differentiation (P = 0.02), and death (P = 0.001). In univariate Cox regression analysis for survival, high GLUT1 expression, presence of any comorbidity, diabetic condition, advanced or metastatic stage, and liver metastasis were significant risk factors for death. CRC patients with high GLUT1 expression showed worse survival outcomes than those with low GLUT1 expression (P = 0.001). Furthermore, the high GLUT1/diabetes (+) patients had an inferior survival outcome than the patients with low GLUT1/diabetes (+) condition.
Conclusions: GLUT1 is significantly upregulated in colorectal adenocarcinoma. The expression correlated with poor tumor histology, higher stage, hepatic metastases, and adverse survival in the study cohort.
Keywords: Colorectal adenocarcinoma, diabetes, glucose transporter 1, real-time polymerase chain reaction assay, tumor metabolism
|How to cite this article:|
Vasudevan S, Mehta A, Sharma SK, Sharma A. Expression of glucose transporter 1 (SLC2A1) – Clinicopathological associations and survival in an Indian cohort of colorectal cancer patients. J Can Res Ther 2022;18:650-5
|How to cite this URL:|
Vasudevan S, Mehta A, Sharma SK, Sharma A. Expression of glucose transporter 1 (SLC2A1) – Clinicopathological associations and survival in an Indian cohort of colorectal cancer patients. J Can Res Ther [serial online] 2022 [cited 2022 Dec 6];18:650-5. Available from: https://www.cancerjournal.net/text.asp?2022/18/3/650/344240
| > Introduction|| |
Cancer cells have a higher metabolic rate than normal cells and switch to aerobic glycolysis from oxidative phosphorylation for energy generation (Warburg effect). Aerobic glycolysis is an inefficient means of producing adenosine triphosphate (ATP) hence, constitutive activation of glucose transport genes maintains an increased glucose flux to meet the energy requirement of neoplastic cells.,
Among the 14 facilitative glucose transporter (GLUT) isoforms (GLUT1-GLUT14), GLUT1 has wide tissue distribution. Encoded by the SLC2A1 gene, GLUT1 is a high-affinity uniporter facilitating the passive uptake of glucose across the plasma membrane. Previous studies suggest that GLUT1 overexpression is a marker of malignant transformation in colorectal cancer (CRC),,, but its role in the CRC prognosis is not well established. Some studies report that GLUT1 overexpression is independently associated with poor prognosis in CRC patients,,,, other studies by Jun et al. and Dai et al. show that it is not an independent predictor of survival., There are no literature reports of GLUT1 expression and survival of CRC patients from India. Furthermore, recent reports suggest that diabetes mellitus is associated with increased risk and inferior outcomes in CRC.,,
This study investigates the GLUT1 transcript expression in a CRC patient cohort and examines the relationship between clinicopathological factors and survival. Further, the effect of GLUT1 expression on survival was assessed depending on the diabetic/nondiabetic condition of the CRC patients.
| > Materials and Methods|| |
Subjects and block selection
The study was approved by the Institutional Review Board (RGCIRC/IRB/235/2018) and conducted in compliance with the Declaration of Helsinki.
Formalin-fixed, paraffin-embedded (FFPE) tissue blocks (tumor and corresponding normal tissue) of ninety upfront surgically resected CRC cases with adenocarcinoma histology, between 2012 and 2017, were identified. Lymph node samples and the patients with more than one cancer or nonprimary CRC were excluded. The patients belonged to the northern parts of India including northeast India.
Tissue sectioning, staining, and real-time polymerase chain reaction
About five sections of 4 μm were cut. Hematoxylin and eosin-stained slides were prepared and manual macrodissection was performed (by an experienced Pathologist, AM) for the tumor sections and adjacent normal tissue sections. Total ribonucleic acid (RNA) was extracted from the marked tissue sections using Promega ReliaPrep FFPE Total RNA kit (Promega, USA) according to the manufacturer's protocol, and quantitated on Qubit® 3.0 Fluorometer (Invitrogen Life Technologies, USA). About 40–50 ng of RNA was reverse transcribed to cDNA by SuperScript VILO cDNA synthesis kit (ThermoFisher Scientific, USA). Primers 5'-CCTGCAGTTTGGCTACAACA-3' (sense) and 5'-GTGGACCCATGTCTGGTTG-3' (antisense) for SLC2A1 and 5'-ACAGAGCCTCGCCTTTGC-3' (sense) and 5'-GCGGCGATATCATCATCC-3' (antisense) for β-actin (normalizing control) were synthesized, keeping amplicon size small (<100 bp). Fluorescent probes: FAM-5'-CCCAGAAGGTGATCGAGGAGTTCT-3'-ZEN-lowa Black Quencher for SLC2A1 and Hex-5'- CACACCCGCCGCCAGCTCAC-3'-ZEN-lowa Black Quencher for β-actin were designed (Integrated DNA Technologies, USA). Real-time polymerase chain reaction (PCR) was carried out using TaqMan Universal Master Mix (Applied Biosystem, USA) on Qiagen Rotor-Gene Q real-time PCR. The thermocycling conditions were 95°C for 10 min (1 cycle), 95°C for 15 s, and 60°C for 1 min (40 cycles). The threshold was set at 0.02 for analysis.
Clinical information including clinical stage (according to American Joint Committee on Cancer, 7th edition guidelines) and treatment details were collected from the hospital medical records. The information on preexisting diabetes was noted by the treating physician and confirmed by the blood glucose report. By referring to hospital checkup visits or through telephonic conversation, the follow-up was maintained until September 24, 2020. The median duration of follow-up was 43 months.
The tumor and normal tissue GLUT1 expression was normalized to the β-actin level by the 2-ΔCt method. The fold change in tumor relative to adjacent normal tissue was computed using 2-ΔΔCt method.
Descriptive statistics were used to summarize the data. The difference in the GLUT1 expression between tumor tissue and normal mucosa tissue was compared by the Wilcoxon matched-pairs signed-rank test. The Kruskal–Wallis test was used for comparing the difference in the relative fold change in GLUT1 expression between tumor differentiation (well, moderate, and poor) and tumor stages. Mann–Whitney U-test compared the relative GLUT1 expression in the patients with and without liver metastasis. We calculated the midspread value (interquartile range, [IQR]) of relative GLUT1 expression and consequently divided the CRC patients into high or low GLUT1 expressing groups. According to the clinical stage, the patients were divided into early (Stage I and II), advanced (Stage III), and metastatic (Stage IV) CRC. Pearson's Chi-square test of association was applied for categorical comparisons. Univariate and multivariate logistic regression analyses were carried out to examine the association between GLUT1 expression and CRC-related factors. Survival analyses were performed by the Kaplan–Meier method and logrank test evaluated the difference between outcomes. Overall survival was defined as the time interval between the date of diagnosis until death or last contact. Further, univariate and multivariate Cox regression analysis was used to analyze the effect of various variables on overall survival. P < 0.05 was considered statistically significant. All the statistical analyses were performed using IBM SPSS Version 23.0 software (SPSS Inc., Chicago, IL, USA) or MedCalc Statistical Software version 19.4.0 (Ostend, Belgium).
| > Results|| |
Of the ninety CRC patients selected, 83 case samples met the quality control. The tissue was not appropriate in seven cases and hence, we excluded them from the analysis. The characteristics of the study group are summarized in [Table 1]. Most tumors were Stage II (38.6%) or Stage III (31.3%). After surgical resection, 41 cases (49.4%) were managed by adjuvant chemotherapy and/or radiotherapy. Forty-two CRC patients (50.6%) selected had diabetes mellitus [Table 1].
|Table 1: Baseline characteristics of the study group (n=83 CRC patients)|
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Quantitative real-time PCR analysis showed that the expression of GLUT1 was significantly increased in CRC tissues in comparison to normal mucosa tissues (0.024 ± 0.056 vs. 0.004 ± 0.005; P < 0.0001). GLUT1 was significantly upregulated in poorly differentiated tumors (2.36 ± 1.08, 7.99 ± 14.51, 24.55 ± 41.57 in well, moderate, and poorly differentiated tumors; P = 0.024). The expression significantly varied with tumor stage (3.64 ± 4.06, 12.91 ± 29.1, 6.48 ± 9.14 and 11.07 ± 8.94 in Stage I, Stage II, Stage III, and Stage IV, respectively; P = 0.045) and was significantly elevated in patients with liver metastasis (11.06 ± 9.01 vs. 9.14 ± 20.71; P = 0.013). The relative fold change of GLUT1 expression in tumor tissue compared with normal mucosa tissue ranged from 1.01 to 133.43 times in the study group. The relative fold change values were divided into quartiles and the IQR of 5.2 fold was taken as the cutoff value to define the GLUT1 high/low expression groups. We selected IQR as the reference to reduce the effect of the outlier values and as a representative figure for GLUT1 expression in the majority of patients in the study group. Twenty-six CRC patients (31.3%) displayed a high GLUT1 expression.
Next, to understand the variables associated with high GLUT1 expression, we compared the high and low GLUT1 expression CRC groups [Table 2]. No significant relationship was observed between the two groups with respect to age groups, gender, tumor location, laterality, and preexisting diabetes [Table 2]. The high GLUT1 expression correlated significantly with advanced and metastatic tumors (P = 0.003), presence of liver metastasis (P = 0.003), and poor tumor differentiation (P = 0.02). Death was more common in the high GLUT1 expressing CRC group than the low GLUT1 expressing group (65.38% vs. 26.32%, P = 0.001) [Table 2]. Univariate analysis showed that high GLUT1 expression was significantly associated with metastatic tumors (odds ratio [OR]: 8.50; [95% confidence interval [CI]: 2.27–31.86]; P = 0.002 for metastatic vs. early stage), the presence of liver metastasis (OR: 7.01; [95% CI: 1.91–25.69]; P = 0.003), poor tumor differentiation (OR: 5.40; [95% CI: 1.23–23.67]; P = 0.025), and survival (OR: 5.28; [95% CI: 1.94–14.38]; P = 0.001) [Table 2]. In the multivariate analysis, high GLUT1 expression was independently associated with poor tumor differentiation (OR: 8.98; [95% CI: 1.40–57.77]; P = 0.028). On univariate Cox regression analysis for overall survival, high GLUT1 expression, the presence of comorbidity, diabetic condition, advanced/metastatic tumor stage, and presence of liver metastasis were statistically significant [Table 3]. In the multivariate Cox regression analysis, only clinical stage was significantly associated with overall survival with worse survival for the patients with metastatic tumors (hazard ratio 11.96, 95% CI [2.15–66.37]; P = 0.004) [Table 3].
|Table 2: Clinical association and logistic regression analysis of the high and low glucose transporter 1 expressing tumor groups|
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The overall survival time for CRC patients with high GLUT1 expression was significantly shorter than the low GLUT1 expression group (P = 0.001) [Figure 1]a. The median overall survival of the high GLUT1 expression group was 44 ± 3.44 months (95% CI: 37.25–50.75). Moreover, the survival outcome of diabetic CRC patients was inferior to the nondiabetic CRC patients (P = 0.036) [Figure 1]b. To understand the impact of GLUT1 expression on survival in hyperglycemic CRC patients, we stratified the GLUT1 expression (high/low) according to the diabetic/nondiabetic state of the patients. The survival was significantly better in the low GLUT1/diabetes (−) group than the low GLUT1/diabetes (+) or high GLUT1/diabetes (+) or high GLUT1/diabetes (−) groups (P = 0.003) [Figure 1]c.
|Figure 1: Kaplan–Meier survival analysis of the study cohort according to (a) High or low glucose transporter 1 expression. (b) Diabetic or nondiabetic status. (c) High or low glucose transporter 1 expression stratified according to diabetic and non-diabetic state (N = 83, colorectal cancer cases)|
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| > Discussion|| |
The prognostic indicators of CRC that have been well explored are tumor stage, differentiation, and serum carcinoembryonic antigen level; however, additional molecular prognostic markers are being studied and they need to be explored in different populations. One of the major hallmark events in cancer is metabolic reprogramming, with glucose used as the chief source of energy. Activation of GLUT1 in cancer cells can be a constitutive response to support increased glucose uptake and glycolysis or an adaptive response to hypoxia in the tumor microenvironment., Previous immunohistochemical studies have suggested GLUT1 overexpression in colorectal adenocarcinoma to be significantly associated with lymph node metastases., Overexpression of GLUT1 supplies glucose to feed the overtly active cancer cells to maintain the high energy required for tumor growth, invasiveness, and metastasis. Other real-time PCR studies by Shen et al. and by Feng et al. found increased GLUT1 messenger RNA (mRNA) expression in primary CRC tumors than the corresponding normal tissue and high GLUT1 expression independently conferred poor prognosis., To the best of our knowledge, ours is the first study examining the association between GLUT1 mRNA expression and CRC in the Indian population. Similar to the previous studies, we observed an increased GLUT1 expression in CRC tissue than in the adjacent normal mucosa. The expression was significantly higher in poorly differentiated tumors than the well/moderately differentiated tumors, and in the patients with hepatic metastasis, indicating that enhanced GLUT1 expression related to poor differentiation and advanced disease.
Regarding stage-wise alterations in the GLUT1 expression, previous studies have shown differing results that could be because of variations in techniques used to detect expression, and selection of the tissue type. Some studies show that expression of GLUT1 is a late event in the CRC genesis,, whereas Shen et al. observed maximal GLUT1 expression in Stage III followed by a decrease in Stage IV, suggesting GLUT1 overexpression as an early and intermediate event in CRC progression. In our study, high GLUT1 expression is associated with advanced and metastatic CRC. Comparable to previous studies, we observed that high GLUT1 expression in CRC tumors correlated with poor differentiation and liver metastasis.,, Furthermore, the survival was inferior in the patients exhibiting high GLUT1 expression or having diabetes. Since patients with diabetes have high ambient glucose levels, it was of interest to study GLUT1 expression in diabetic CRC patients. Survival analysis showed that the high GLUT1/diabetes (+) group had an inferior survival outcome than the patients with low GLUT1/diabetes (+) group indicating a cumulative effect of adverse events or a common induction of oncogenic signaling. We found no association of high GLUT1 expression with diabetes which could possibly be because GLUT1 overexpression is caused by mechanisms other than hyperinsulinemia associated with maturity-onset diabetes. It is established that GLUT4 is the canonical insulin-responsive transporter found in muscle and adipocytes, and GLUT1 has ubiquitous distribution and mediates basal transport of glucose in an insulin-independent manner. Oncogenes such as RAS, reactive oxygen species, and hypoxia-inducible factor 1-alpha (HIF1α) transcriptionally regulate GLUT1 and are also implicated in the development of diabetic complications.,, The stabilization of the transcription factor HIF1α under tumor hypoxic conditions stimulates glycolytic genes including GLUT1.,
| > Conclusions|| |
The data suggest that CRC tumor tissue significantly overexpress GLUT1 than the adjacent normal mucosa. High GLUT1 expression significantly correlates with poor tumor histology, higher stage, hepatic metastases, and adverse survival in the study cohort.
We express our thanks to Mr. Ajay and Mr. Tulsi Ram, Histopathology Department, for assisting in the procurement of FFPE blocks, tissue sectioning, and hematoxylin and eosin staining.
The authors thank Mr. Surender Danda, Molecular Diagnostics, for confirming the GLUT1 and β-actin primers by Sanger sequencing.
Financial support and sponsorship
The study was financially supported by the Rajiv Gandhi Cancer Institute and Research Centre -Scientific Committee Research Grant (Res/SCM/28/2018/37).
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]