Validity and clinical impact of glucose transporter 1 expression in colorectal cancer.

BACKGROUND/AIM: There is no doubt that colorectal cancer (CRC) poses a major threat to public health worldwide, and despite improvement in managements, prognosis still remains an irritating question with no definite answer. Being a fundamental player in cancer metabolism, glucose transporter 1 (GLUT1) could be utilized as a prognostic biomarker that could fuel development of new treatment strategies. The aim of this study was to assess the validity of GLUT1 expression as a prognostic biomarker and to elucidate to what extent it is immersed in poor clinical outcome among CRC patients. PATIENTS AND METHODS: GLUT1 expression in peripheral blood specimens was analyzed by quantitative real-time polymerase chain reaction in 47 CRC patients and 20 healthy controls.
RESULTS: There was significantly elevated GLUT1 expression in peripheral blood of CRC patients than in controls (P < 0.001). The cutoff value of 0.605 provided 98% sensitivity and 100% specificity. There were significantly higher values of GLUT1 expression in patients under 50 years (P = 0.003), performance status 2 (P = 0.009), stage IV (P < 0.001), and presence of metastasis (P < 0.001). GLUT1 expression showed nonsignificant association with overall survival (P = 0.068), while tumor stage (P = 0.01) and metastasis (P = 0.009) were significantly associated with lower overall survival.
CONCLUSION: GLUT1 is sensitive and specific marker for CRC. It is overexpressed in young age patients, poor performance status, and stage IV patients. Although this was not statistically significant, GLUT 1 showed higher expression level in patients with lesser survival.

INTRODUCTION

Colorectal cancer (CRC), the third most common cancer and the fourth leading cause of cancer-related death worldwide,[1] poses a major burden to public health.[2345] In Egypt, CRC represents 4.2% of the total tumor burden. It is ranked fourth in females and seventh in males. According to National Cancer Institute Cairo records, it is more common in males, and the median age in Egyptian patients is about 50 years.[6] Identification of prognostic biomarkers that could fuel development of new treatment strategies would be of a particular clinical relevance.[78]

Reprogramed glucose metabolism is one of the cornerstones of cancer homeostasis.[910111213] In comparison to normally differentiated cells, CRC cells uptake glucose at a higher rate to feed the highly active aerobic glycolysis.[481014] This enhanced uptake is mediated by glucose transporter 1 (GLUT1).[1516171819]

GLUT1, encoded by the SLC2A1 gene, belongs to the facilitative superfamily of membrane integral proteins, a family of 14 members.[2021222324] GLUT1 is the major glucose transporter in CRC cells.[16252627] It is formed of 492 amino acid residues and possesses a single site of N-linked glycosylation at N45.[2028]

Cancer cells modulate glucose uptake, the first step in glucose metabolism, by induction of GLUT1.[11] Previous studies have reported GLUT1 overexpression in various tumors;[782930313233343536373839404142434445] however, there was a little consistency in results that were presented. Furthermore, considerable differences in methodological approach have prevented the reliable comparisons necessary to determine the true clinical value of GLUT1 gene expression in peripheral blood of CRC patients. In a metastatic state, each gram of tumor may shed approximately 106 cells into the blood vessel.[46] Being easy and safe to perform, blood tests is a good procedure.[39] In contrast, analysis of solid tumors necessitates invasive procedures that might limit patient compliance. Thus identification and validation of prognostic biomarkers of peripheral circulating cancer cells in blood specimens could be of great help in terms of patient compliance. We exploited real-time polymerase chain reaction (RT-PCR) to assess the validity of GLUT1 expression in CRC peripheral blood specimens and to explore to what extent this expression profile is related to clinical features as well as to overall survival of CRC patients.

PATIENTS AND METHODS

This case–control study was carried out on patients with histopathological proof of colorectal adenocarcinoma who attended to new cases clinic in Clinical Oncology Department, Faculty of Medicine, Menoufia University, Shibin El Kom, Egypt. In the study period from March 2014 to October 2014, a total of 47 patients were enrolled; out of which 35 patients diagnosed with colon adenocarcinoma and 12 patients diagnosed with rectal adenocarcinoma. Twenty age and gender-matched healthy subjects were included as a control group.

A simple and clear explanation of the research objectives and procedures was provided to each of the controls in the study. All patients were subjected to full history taking (including age, gender, complaint, comorbidities, family and personal history of cancer and surgical interference), thorough clinical examination (including weight, height, performance status, local and general examination), and full investigations [body computed tomography (CT), colonoscopy, complete blood count, full kidney and liver functions].

Ethical approval was obtained from the Research Ethics Committee, Faculty of Medicine, Menoufia University, Shibin El Kom El-Kom, Egypt and informed consent was obtained from every participant. Both patients and controls were subjected to the analysis of mRNA expression levels of GLUT1 by RT-PCR.

Gene expression analysis of GLUT1

RNA isolation

Total RNA was extracted from whole blood (collected in ethylenediamine tetraacetic acid tube) by GeneJET Whole Blood RNA Purification Mini Kit (Thermo Scientific), according to the manufacturers' protocol. RNA samples were stored at –20°C until analysis. The concentration of RNA was determined by measuring its absorbance at 260 nm (A260). Absorbance readings should be >0.15 to ensure significance. The ratio between the absorbance value at 260 and 280 nm (A260:A280) gives an estimate of RNA purity. (A260:A280) ratio >1.6 was accepted. Two-step RT-PCR was done as follows: for reverse transcription step, a reverse transcriptase kit (SensiFAST cDNA synthesis kit, Bioline Reagents Ltd, UK) was used for complementary DNA (cDNA) synthesis on 2720 thermal cycler (Singapore). For cDNA synthesis, RNA (10 μl) was reverse transcribed in a final volume of 20 μl containing 1 μl of reverse transcriptase enzyme, 4 μl of 5x TransAmp buffer, and 5 μl of DNase/RNase free water. The samples were incubated at 25°C for 10 min (primer annealing), and 42°C for 15 min (reverse transcription). Reverse transcriptase was then inactivated by heating at 85°C for 5 min. All products were stored at −20°C till the next step. For cDNA amplification, a relative quantitation of GLUT1 mRNA expression normalized to the endogenous reference gene β-actin was performed by RT-PCR reverse transcription, using the 2x SensiFAST™ SYBR® Lo ROX Kit (Bioline Reagents Ltd. located in Humber Road, The Edge Business Centre, Unit 16, London, NW2 6EWUnited Kingdom), on Applied Biosystems 7500 RTPCR system. GLUT1 primers were: 5′-CAACTGGACCTCAAATTTCATTGTGGG-3′ (forward) and 5′-CGGGTGTCTTATCA CTTTGGCTGG-3′ (reverse).[47] β-actin was used as an endogenous reference with primers: 5′-AGTTGCGTTACACCCTTTCTTG-3′ (forward) and 5′-TCACCTTCACCGTTCCAGTTT-3′ (reverse). Specificity of the primers was verified using Primer BLAST program provided by NCBI. The PCR reaction mixture (final volume, 25 μl) contained 12.5 μl of 2x SensiFAST™ SYBR® Lo ROX Master Mix, 1 μl of each primer (Sigma), 5.5 μl of DNase/RNase free water, and 5 μl of cDNA. Thermocycling conditions were 10 min at 95°C, followed by 45 cycles at 95°C for 15 s, and 60°C for 1 min. For relative quantification of the results obtained by RT-PCR, the comparative cycle threshold (Ct) method was used. Analysis was performed using Applied Biosystems 7500, software version 2.0.1.

Patients were followed up for at least 24 months by CT scan for abdomen and pelvis (searching for liver metastases), tumor markers (CA19-9 and CEA) every 3 months, and annual colonoscopy for cases of colon cancer, whereas rectal cancer patients were followed up by magnetic resonance imaging of pelvis (searching for local recurrence), tumor markers (CA19-9 and CEA) every 3 months, and annual colonoscopy.

Time to progression was calculated for all patients as the length of time from the date of start of treatment until the disease starts to progress in the form of local recurrence, newly developed metastases (for patients with localized disease), or increase in size and/or number of metastases in patients with primary metastatic disease. Overall survival was calculated as the length of time from the date of start of treatment until date of patient's death.

Statistical analysis

The Statistical Package for the Social Sciences version 16 (SPSS Inc., Chicago, IL, USA) was used in data analysis. Descriptive statistics were used to present the distribution of demographic and clinical characteristics. The Chi-square and Fisher's exact tests were used for qualitative data. t-test, Mann–Whitney, and Kruskal–Wallis tests were used to test the difference in quantitative data. The odds ratio (OR) and 95% confidence intervals (CI) were calculated. P value <0.05 was considered to be statistically significant. Response to treatment was assessed according to revised RECIST guideline (version 1.1).[48] Receiver operating characteristics (ROC) curve was used to assess sensitivity, specificity, and to determine cutoff point. Survival was analyzed using the Kaplan–Meier curve.

RESULTS

As shown in Table 1, out of the 47 patients, 35 (74.5%) patients were diagnosed with colon adenocarcinoma, while only 12 (25.5%) patients were diagnosed with rectal adenocarcinoma. The mean age of patients was 50.25 ± 10.79 years. The patients under 50 years constituted 53.1% of all CRC patients. There were 20 (42.6%) males versus 27 (57.4%) females. The mean ± SD of BMI was 25.98 ± 5.22 within the patients. Abdominal pain and bleeding per rectum were the most common presenting symptoms (51.1 and 19.1%, respectively). Out of all patients, 26 patients (61.7%) were of performance status 0, 15 patients (31.9%) were of performance status 1, and only 3 patients (6.4%) with performance status 2. Also most of patients were nonsmokers (83.0%) and had no comorbidities (57.4%).

Table 1

Descriptive statistics of the different parameters in (CRC) patients

Regarding the location of tumor, the most common site was the right colon (36.2%) followed by left colon, upper rectum (tumor lies >9 cm up to 19 cm from anal verge, i.e. in the upper two-thirds of the rectum), lower rectum (tumor lies within 9 cm from the anal verge, i.e. in the lower third of the rectum), and transverse colon (31.9, 14.9% and 10.6, 6.4%, respectively).

Most patients were in stage III and IV representing 40.4 and 44.7%, respectively. Interestingly, the liver was the most frequently involved site either alone or in association with other involved sites. The majority of patients had moderately or poorly differentiated histologic tumor types (72.3 and 23.4%, respectively); only one patient (2.1%) had signet ring differentiation, while 37 (78.7%) patients showed non-mucinous differentiation.

Twenty-eight patients representing 59.6% were able to undergo surgery as primary treatment modality. During follow-up most patients completed their treatment schedule (either with chemotherapy or concomitant chemo and radiotherapy) with no treatment-related toxicities (61.7%). At the end of follow-up period, 25 (53.2%) patients died, while 22 (46.8%) survived with overall survival rate of 46.8%.

Based on RT-PCR data, there was significantly elevated GLUT1 expression in peripheral blood of the 47 CRC patients in comparison to the 20 healthy controls (P < 0.001) [Table 2 and Figure 1].

Table 2

GLUT1 expression among the studied subjects

Figure 1

Amplification plot of GLUT1 expression (ΔRn vs. Cycle)

To evaluate the diagnostic power of the quantitative GLUT1 assay to discriminate CRC from healthy individuals, ROC curve analysis was performed. The cutoff value of 0.605 provided 98% sensitivity and 100% specificity (area under the curve was 0.98, suggestive of a high discrimination power, positive predictive value 100%, negative predictive value 95%) [Figure 2 and Table 3].

Figure 2

ROC curve of GLUT1 to differentiate between cases and control

Table 3

Validity of GLUT1 expression in diagnosis of malignant transformation

The association between GLUT1 expression and different clinico-pathologic prognostic parameters is shown in Table 4 in which there was statistically significant relation between GLUT1 expression and age, performance status, tumor stage and metastasis with higher values in patients under 50 years (P = 0.003), performance status 2 (P = 0.009), stage IV (P < 0.001), and presence of metastasis (P < 0.001). There was no significant difference in GLUT1 expression regarding gender (P = 0.788), tumor location (P = 0.372), tumor differentiation (P = 0.878), and initial tumor markers level [Table 4 and Figure 3].

Table 4

GLUT1 expression in relation to different clinicopathologic prognostic variables

Figure 3

Overall survival among the studied cases

Moreover, there was an elevated GLUT1 expression level in died patients compared to survived ones, but this finding did not reach a significant level (P = 0.068) [Table 4 and Figure 4]. There was significant correlation between GLUT1 expression and both of age (P = 0.017) and tumor stage (P ≤ 0.001), while there was no significant correlation between GLUT1 expression and either BMI (P = 0.074), grade (P = 0.710), performance status (P = 0.425), or overall survival (P = 0.128) [Table 5].

Figure 4

GLUT1 and survival analysis

Table 5

Correlation between marker results and some chosen parameters

Regarding survival analysis, the median overall survival was 24 months [Table 6]. By univariate analysis, tumor stage (P = 0.01) and metastasis (P = 0.009) were significantly associated with lower overall survival [Table 7 and Figures 5, 6], while other factors such as age, gender, performance status, tumor differentiation were nonsignificant predictive factors [Table 7].

Table 6

Mean and median overall survival among the CRC patients

Table 7

Univariate analysis for factors affecting overall survival among the CRC patients

Figure 5

Tumor stage and survival analysis

Figure 6

Metastasis and survival analysis

DISCUSSION

In order to proliferate, enhanced glycolytic profile is a constitutive tumor survival response.[1149] GLUT1 plays a fundamental role in cancer metabolism.[450] Expressing high levels of the GLUT1 is a cancer tool to resist the harsh tumor microenvironment.[4101439]

Accumulating evidence has demonstrated GLUT1 overexpression in a wide variety of tumors obtained with different methodologies.[25] However, there are few data confirming this metabolic phenotype by sensitive and relatively an easy method. Accordingly, we exploited RT-PCR technique to clarify the validity and prognostic significance of GLUT1 expression in peripheral blood of CRC patients and its association with patient outcome and overall survival.

In this prospective study, patients under 50 years constituted 53.1% of all CRC patients. This is higher than previous a study in Egypt in which the incidence of patients under 50 years was 32%,[8] indicating that CRC incidence in patients under 50 years in Egypt is increasing. The current data showed a higher female prevalence (57.4%). This is slightly different from Rashed et al.[8] and Eisa studies[51] (40 and 41.6%, respectively).

According to our results, the most common site was the right colon, while the rectum represented 25.5%. This is similar to an earlier Egyptian publication where the rectum constituted 27%.[52] Conversely, in a study by Eisa, rectal carcinoma constituted 42.7%.[51]

The present findings revealed that most patients were in stage III and IV (40.4 and 44.7%, respectively) and 44.7% of all patients had metastatic lesions. In concordance with our results, Eisa observed that young patients had more advanced stage at presentation and explained this either due to the aggressive behavior of the tumor itself or delay in diagnosis.[51]

In this study there was a significant elevated GLUT1 expression in peripheral blood of CRC patients in comparison to controls. Interestingly, the discriminative power of GLUT1 expression was 98% sensitivity and 100% specificity. This exceeds what was documented in a previous report about other serological markers used in CRC such as CEA and CA19-9 where their sensitivity was only 30% and 18%, respectively.[53] This confirmed that GLUT1 is a reliable diagnostic marker for tumorigenesis and may be used as an indicator of possible malignant transformation in high-risk patients such as those with multiple polyposis and ulcerative colitis.

CRC, as many solid tumors, experiences hypoxic microenvironment.[405455] Hypoxia-inducible factor-1, when stabilized by hypoxia, upregulates several glycolytic genes to promote survival under these tough environments.[56] One of these genes is GLUT1 gene.[3957585960] It is likely that CRC cells upregulate GLUT1, thus increasing glucose uptake[4] to feed enhanced glycolysis as they require high energy levels to proliferate.[255561] Moreover, GLUT1 expression is suppressed by p53, an important tumor suppressor in cancer.[62] The alteration in p53 expression may explain GLUT1 overexpression observed in many cancer types, as well as their enhanced glucose metabolism and their higher energy consumption.[16]

Similar to the current observation, Chung et al.[39] found that GLUT1 mRNA was increased in the peripheral blood of stage II and III CRC patients as compared to stage I patients, suggesting that GLUT1 may be a stage-related marker that could be determined by a noninvasive method.

This study confirmed the existence of significantly higher values of GLUT1 expression in patients under 50 years, performance status 2, stage IV, and presence of metastasis. This reflected the close relation between GLUT1 overexpression and poor clinicopathologic factors representing a warning sign of aggressive tumor behavior. High levels of GLUT1 expression make cancer cells resistant to a hypoglycemic environment and have the propensity to survive, proliferate, and metastasize.[14] This means that these patients will benefit from GLUT1 inhibitors. Similar to our finding, Younes et al., Haber et al., Sakashita et al., and Chung et al. noticed that induction of GLUT1 is significantly associated with lymph node metastasis and poor prognosis in CRC.[30313539]

Previous studies based on immune-histochemical detection of GLUT1 also showed that GLUT1 overexpression was an indicator of poor prognostic parameters in CRC.[478313235]

This study showed that there was a lack of significant association between GLUT1 expression and gender, tumor site, and tumor differentiation. Similarly, Younes et al.[30] and Haber et al.[31] reported that there was no correlation between GLUT1 expression and histologic differentiation. Moreover, there was no significant difference in GLUT1 expression regarding the initial level of CA19-9 and CEA.

Currently, there was significant correlation between GLUT1 expression and both of age and tumor stage, while there was no statistically significant relation between GLUT1 expression and overall survival. Consistently, the pooled data gathered by Yang et al demonstrated that GLUT1 still had no significant association with overall survival irrespective of tumor location, cancer type, and treatment.[55] On the contrary, Jun et al.[7] documented that patients with GLUT1 expression demonstrated poor overall survival and disease-free survival.

In the present study, tumor stage and presence of metastases had a statistically significant relation with overall survival, with the least survival in advanced stage and metastatic disease. In agreement with this, Eisa stated that stage at presentation, lymph node involvement, and performance status are predictors for overall survival in young CRC patients.[51]

CONCLUSION

Taken as a whole, these results support the fundamental role played by GLUT1 in tumor growth and progression, making it a potential biomarker of tumor detection and patient prognosis. Also, this study sheds light on exploitation of this technique on peripheral blood samples maximizing patient compliance. However, approval of application of this technique on peripheral blood samples of CRC patient needs further research allowing easy, sensitive, and most importantly, repeated detection of GLUT1 for early detection and proper therapeutic interventions. Overall, GLUT1 should be targeted in addition to other traditional therapeutic lines.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.