Increased expression of Glut is detected in

Increased expression of Glut1 is detected in a number of cancer types including lung [7], brain [8], bladder [9], cervical [10], colorectal [11], esophageal [12], hepatocellular [13], head and neck [14], gastric [15], ovarian [16], renal cell [17], pancreatic [18], thyroid [19] and breast [20]. Overexpression of Glut1 is associated with a poor prognosis in several cancers including those of the breast [20], [21]. Therefore, Glut1 represents a potential therapeutic target for many cancers. Recent studies have focused on inhibiting Gluts to decrease cancer cell growth [22], [23], [24], [25]. Basal-like breast cancers generally lack estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2) on clinical assays and commonly referred to as “triple-negative” [26]. TNBCs typically have poor prognosis due to aggressive tumor phenotypes. Conventional targeted therapies cannot be applied to TNBCs, leaving chemotherapy as the only option [27], [28]. Glut1 expression is significantly increased in basal-like breast cancer and is regarded as a potential therapeutic target [29].
Materials and methods
Results
Discussion Glut1 is detected at an elevated level in cancers and is considered as a potential therapeutic target [29]. One therapeutic approach is the use of antisense oligonucleotides against Glut1 genes. Amannet al. proposed Glut1 as a therapeutic target for hepatocellular carcinoma (HCC) [13]. They showed that inhibition of Glut1 in HCC cells by siRNA decreased cell proliferation and migration. However, they did not assess apoptotic cell death by Glut1 siRNA. Another therapeutic strategy against cancer cells is Glut1 antibodies. Rastogi et al. observed that BRL 37344, sodium salt against Glut1 induce growth arrest and apoptosis in human breast cancer and lung cancer cell lines [25]. They observed that Glut1 antibodies decrease proliferation and increase chemotherapy-induced apoptosis. To examine the possibility of Glut1 as a therapeutic target in TNBC cells, we established Glut1 knockdown cell lines in MDA-MB-231 and Hs578T cells. We previously found that silencing Glut1 decreases cell proliferation, glyclolytic phenotype, migration and invasion [59]. The results suggest that Glut1 might be a therapeutic target for TNBCs. In this report, we focused on the Glut1 silencing effect on cell viability since the ideal tumor response to targeted therapy is apoptotic cell death. We hypothesized that Glut1 ablation would reduce apoptosis and render cells more sensitive to treatment with chemotherapeutic agents. Contrary to our hypothesis, silencing of Glut1 attenuated apoptosis and induced chemoresistance (Fig. 2). In Glut1 shRNA cells, decreased cleavage of caspase-3 was observed after doxorubicin, gemcitabine, irinotecan, etoposide or paclitaxel treatment (Fig. 4A and B). Decreased cleavage of caspase-3 was due to upregulation of survivin in Glut1 shRNA cells. Survivin (BIRC5) is a member of the inhibitor-of-apoptosis proteins family and decreases the activity of caspase-3 and induces resistance to apoptosis [46], [47]. BIRC5 transcription is upregulated by β-catenin [50], [51], [52]. We found that expression of survivin and β-catenin was elevated in Glut1 shRNA cells even in the presence of drugs (Fig. 4). β-catenin is negatively regulated by GSK-3β activity at the translational level. GSK-3β phosphorylates β-catenin, culminating in the ubiquitination and degradation of β-catenin [53]. We found that phosphorylation of GSK-3β at Ser9 to an inactive form increased in Glut1 shRNA cells (Fig. 3A), suggesting a decrease in the active form of GSK-3β. This increased phosphorylation of GSK-3β resulted from upregulation of Akt in Glut1 shRNA cells (Fig. 3, Fig. 5B). Akt activity was significantly increased despite reduced EGFR activity (Fig. 3A). This was because PTEN levels decreased via an EGFR/ERK/Egr-1 pathway. PTEN is a negative regulator of the PI3K/Akt pathway [40]. In addition to EGFR phosphorylation, Glut1 silencing decreased EGFR mRNA and protein (Fig. 3A and B). Although Glut1 and EGFR physically interact, we concluded that Glut1 could not regulate EGFR protein stability (Fig. 7B, C and 7D). Instead, we assumed that Glut1 may regulate EGFR transcription via Egr-1. Egr-1 is a transcription factor of EGFR [57], [58]. Currently, we do not have an explanation for how Glut1-silencing induced downregulation of EGFR transcription. We also could not completely rule out the involvement of an unknown signaling mechanism between Glut1 and EGFR/ERK/Egr-1 signaling.