Expression of Facilitative Glucose Transporters in Cancer
October 1, 1999 9 minutes read
Expression of Facilitative Glucose Transporters in Cancer
By Yoshikam Noguchi, MD, FACS; Takaki Yoshikawa, MD, PhD; Doulet Marat, MD, PhD; and Akira Tosubaraya, MD, DMSci
Increased glucose uptake seems to be one of the major metabolic alterations observed in malignant tissues. Tumor cells may acquire the expression of facilitative glucose transporter (GLUT)1 mRNA during the process of malignant transformation. In some tissues, including stomach, colon, and lung, GLUT1 mRNA is not present in the normal epithelium but is detected in corresponding malignant tissues from the same organs. Increased GLUT1 expression may be one possible mechanism for increased uptake of [18F] fluorodeoxy glucose in positron emission tomography (PET). It also has been suggested that overexpression of GLUT1 protein is associated with poor prognosis in patients with gastric cancer or breast cancer. Thus, facilitative glucose transporters, especially GLUT1, may be good candidates to serve as molecular targets both as clinical prognosticators and as the basis for the development of future therapy. This article reviews some aspects of GLUT related to cancer and presents some of our data on gastric and lung cancers.
Glucose Uptake into Cells
Cancer cells have increased rates of glucose metabolism compared with normal cells.1 There are two types of glucose carriers in mammalian cells,2 Na+-glucose co-transporters and facilitative GLUTs. GLUTs in the plasma membrane are responsible for facilitating glucose flux between blood and tissue. It has been established that glucose transport is the rate-limiting step in glucose utilization in some tissues. A family of seven facilitative GLUTs (GLUT1-GLUT7) has been cloned. GLUT1 is expressed in erythrocytes and is known as a basic, high-affinity glucose transporter. GLUT2 is predominantly expressed in the liver and GLUT3 in the brain. GLUT4 is abundant in insulin-responsive tissue (i.e., adipose and muscle) and is known as the insulin-responsive glucose transporter. GLUT5 was recently determined to be a fructose transporter and is often excluded from the glucose transporter family. GLUT6 appears to be not translated, and GLUT7 is the microsomal glucose transporter.
Recently, data on the expression or significance of these transporters in tumors and the impact this may have on the host have been accumulated. Also, the clinical application of the PET scan, which utilizes [18F] fluorodeoxy glucose as a tracer to detect presence of tumor and its metastases, has provoked interest in the mechanisms by which tumors aggressively increase glucose uptake over the tumor-bearing hosts.3 Furthermore, the increased expression of GLUTs in some tumors has been associated with poorer prognosis in various malignancies.
Expression of GLUT Isoforms in Cancer Tissues
In a study of GLUT expression in breast cancer using immunohistochemistry,4 GLUT1 and GLUT2 expression was identified in all primary tumors and metastatic lymph nodes tested. Expression of GLUT4 was detected in 50% of cases, and GLUT3 and GLUT5 was detected in none of the samples. Staining of normal mammary epithelium for GLUT1, if present, was much weaker than observed in tumor cells from the same patients. In a study on extracranial head and neck tumors, all 20 tumors examined expressed GLUT1 and/or GLUT3 mRNA, leading the authors to conclude that the high-affinity GLUT isoforms may be related to tumor growth, especially when glucose was limiting (e.g., in a poorly vascularized location).5 Only two studies other than ours have provided data on the expression of glucose transporters in gastric cancer. After northern analysis of RNA from one normal tissue sample and two gastric cancer specimens, Yamamoto et al concluded that GLUT1 and GLUT3 were present in tumors to a greater extent than in normal gastric tissue.6 Younes and associates reported GLUT1 expression in three of six samples of adenocarcinoma of the stomach.7
In our study, RT-PCR did not find GLUT1 mRNA in the normal gastric mucosa (n = 10). However, 95% of the gastric cancer specimens we examined (n = 20) contained detectable GLUT1 mRNA. This was also the case with colon cancer and normal mucosa. GLUT1 mRNA was only detected in cancer cells.8 Studies of lung carcinoma and alveolar epithelium yielded identical findings.9 Thus, tumors seem to acquire GLUT1 mRNA and protein by malignant transformation. Whether this hypothesis is generally applicable to all tumors or not has to be further examined.
GLUT1
GLUT1 has a high affinity for glucose and shows an asymmetrical transport (i.e., preferentially in the direction of cellular glucose uptake). Expression of this facilitative GLUT1 is compatible with high metabolic rates in tissues and with regulatory activities such as glucose reabsorption activity and barrier functions. Changes in both GLUT1 expression and the rates of glucose transport have been affected not only by cellular growth rates but by transformation and malignancy. Experimental studies have shown that malignant transformation of cultured cells by either viruses or oncogenes resulted in increased rates of glucose transport, and metabolism.
Expression of GLUT1 was a significant factor affecting survival in the gastric cancer. This accords with a recent report on GLUT1 expression in colorectal cancer. As in colorectal cancer, postoperative survival of patients whose tumor expressed GLUT1 protein was significantly poorer than those with tumors not expressing GLUT1. Thus the high affinity and directional glucose transport of GLUT1 would favor tumor growth at the expense of the tumor-bearing host.
Other GLUTs
GLUT2 expression has been associated with low glucose concentration. The concomitant expression of GLUT1 and GLUT2 mRNA in gastric cancer may be due to a cellular adaptation, a cellular differentiation within the tumor, or a topographical mixture of tissues with different glucose supplies. Clinical singificance of other GLUTs, including 2, 3, and 5, awaits further clarification.
GLUT4 in the Tumor and Insulin Resistance at the Peripheral Tissue in Patients with Cancer
Of GLUT isoforms, GLUT4 is known as the insulin-responsive glucose transporter and is usually found in insulin-responsive tissues such as skeletal muscle and adipose. GLUT4 has been extensively studied as a major cause of non-insulin dependent diabetes mellitus. The presence of GLUT4 mRNA or protein in tumor tissue has been relatively rare, with the exception of breast cancer. However, GLUT4 was the most frequently demonstrated GLUT protein in the gastric cancer specimens. The presence of GLUT4 in human gastric cancer cell lines is in significant contrast to its absence in the Caco-2 colon adenocarcinoma cell line. The presence of the insulin-responsive transporter in cancer tissues may have significant meaning for cancer patients, who are often insulin resistant. In peripheral insulin resistance, the defect is at the level of cellular uptake, not in insulin production. As serum insulin concentration remains elevated and insulin is an anabolic hormone, it has been suggested that this condition could stimulate tumor growth because infused glucose is less effectively taken up by peripheral tissues (mainly muscle). This glucose may be rapidly taken up by cancer cells that express insulin-responsive GLUT4. However, it is not clear whether GLUT4 in tumor cells responds to insulin in the same way that it does in adipose and muscle or what role is played by GLUT4 in the tumor.
GLUTs in Gastric Cancer8,10
The presence of mRNA for five facilitative GLUT isoforms was evaluated by RT-PCR in paired samples of normal gastric mucosa and gastric tumors from 20 individuals. Expression of GLUT proteins was immunohistochemically determined in 70 resected gastric cancer specimens. By RT-PCR, GLUT2 mRNA was detected in 80% of normal gastric mucosal samples, while GLUT4 mRNA was seen in only 40%. GLUT1 mRNA was not detected in normal gastric mucosa. In gastric carcinoma samples, GLUT1 mRNA was detected in 19 out of 20 cases (95%) and GLUT2, GLUT3, and GLUT4 mRNAs in all samples. By immunohistochemistry, GLUT1 protein was detected in 19% of the tumors. A majority of tumors (61%) expressed one or more transporter protein. The presence of GLUT1 protein in a tumor was positively correlated with the tumor’s invasion into the gastric wall, lymphatics, or blood vessels and with lymph node metastases. GLUT1 was a significant factor affecting survival. The postoperative survival of patients with tumor-expressing GLUT1 protein was significantly worse than those with tumors that did not express GLUT1. We concluded that gastric cancer cells may acquire the ability to produce GLUT1 mRNA by malignant transformation. Increased expression of the high affinity glucose transporters, GLUT1 and/or GLUT4, in tumor cells may drain glucose preferentially to the tumor at the expense of the tumor-bearing host.
We also documented the presence of multiple GLUT isoform and response to insulin in human gastric cancer cell lines, MKN28, MKN45, and STSA. RT-PCR demonstrated GLUT1 and GLUT4 mRNA in all three cell lines. MKN28 cells expressed GLUT4 protein more than MKN45 and STSA cells by immunohistochemistry. Insulin stimulation of MKN28 cells resulted in a 22% increase in glucose uptake over that found under basal conditions (0.60 ± 0.05 fmol/cell/min after insulin stimulation vs 0.53 ± 0.07 fmol/cell/3 min at basal). No increase in glucose uptake occurred with insulin stimulation in MKN45 or STSA cells. We conclude that the insulin responsive GLUT4 is expressed in MKN28, MKN45, and STKM1 human gastric cancer cell lines, albeit in different amounts. The greater expression of this transporter in MKN28 cells is likely responsible for the cell’s ability to increase glucose uptake with insulin stimulation. However, a role played by GLUT4 in regulating the amount of glucose uptake would not be large in those human gastric cancer cell lines.
GLUTs in Lung Cancer
Expression of facilitative GLUT isoforms was studied immunohistochemically in lung carcinomas.9 GLUT1 was expressed in 45 (74%) of 61 lung carcinomas, including 19 (100%) of 19 squamous cell carcinomas. No GLUT1 staining was seen in normal lung epithelium surrounding the tumors. In squamous cell carcinomas and small cell carcinomas, GLUT1 immunostaining was stronger in the central area of tumor cell nests corresponding to the hypoperfused region. Focal staining was seen in 14 (58%) of 24 adenocarcinomas, and positive staining was correlated with decreased differentiation, larger tumor size, and positive lymph node metastasis. Glut2 was detected in normal airway epithelium, but no positive staining was seen in lung carcinomas. GLUT3 and GLUT4 were not positively stained in normal lung epithelium, but a few lung carcinoma samples showed positive reaction (9 of 61 in GLUT3; 4 of 61 in GLUT4). GLUT4 immunoexpression was seen in regenerating alveolar and bronchiolar epithelia both around and within cancer cells. GLUT5 expression was not detected in normal or malignant tissue. RT-PCR for GLUT1, GLUT3, and GLUT4 confirmed the expression revealed by immunohistochemical analysis. Overexpression of GLUT could enhance uptake of glucose into lung carcinoma cells, and the increased glucose flux could be involved in cell biologic activities.
PET Scan: New Functional Imaging of Cancer
The availability of PET scanning, which utilizes the tumor’s increased glucose uptake to accumulate [18F] fluorodeoxy glucose (FDG) to a greater extent than in normal tissue, has peaked interest among clinicians in the mechanisms of glucose uptake in human tumors.3 Only recently have detailed data become available on the accelerated glucose uptake that occurs in some tumor tissues. FDG is a glucose analog that is accumulated and subsequently becomes trapped in metabolically active cells. FDG is phosphorylated by hexokinase to FDG 6-phosphate. However, unlike glucose 6-phosphate, FDG 6-phosphate is not further metabolized by glycolytic or glycogen synthetic pathway. Thus, FDG localization within cells depends on the combination of a functional glucose transporter and phosphorylating enzymes. The introduction of PET opened a new era of evaluating the physiology and biochemistry of tumor, whereas imaging modalities such as CT and magnetic resonance imaging (MRI) evaluate alterations of anatomic imaging.
Overexpresion of GLUT1 and increased FDG uptake in pancreatic carcinoma was documented by Reske and coworkers.11 The standardized uptake value (SUV) of FDG in patients with pancreatic cancer was more than twice the SUV of those with chronic mass-forming pancreatitis (MFP) (2.98+1.23 and 1.25+0.51, respectively [P < 0.01]). GLUT1 expression was documented by northern blot analysis in four of five patients with cancer but not in any of the five patients with MFP. The authors concluded that the concomitant enhancement of glucose utilization and selective overexpression of GLUT1 mRNA in pancreatic cancer but not in MFP suggested constitutive activation of GLUT1 gene or decreased degradation of GLUT1 mRNA in human pancreatic cancer.
Commentary
We are currently working on clarifying the clinical significance of GLUT expression in gastric cancer cell lines. Preliminary data demonstrated that suppression of GLUT1 expression was related to "slowing down" the cell cycle by G1 arrest through down regulation of p21 protein and that, as a result, tumor growth was significantly suppressed (presented at AACR in 1999). Further dissection of a classic hypothesis that tumors are "glucose eaters" may provide us with a new target for the early detection and therapy aimed at leading tumors to starvation. (Dr. Noguchi, Dr. Marat, and Dr. Yoshikawa are in First Department of Surgery, Yokohama City University School of Medicine; and Dr. Tsubaraya is in Division of Gastric Surgery, Kanagawa Cancer Center, Yokohama, Japan.)
References
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