Background
Altered metabolism is a characteristic of most cancer cells and contributes to the needs of transformed cells for rapid proliferation, enhanced energy turnover, anabolic pathways and regulation of redox potential [
1,
2]. Both normal and tumor cells use glucose and glutamine as substrates for ATP synthesis and production of components of cellular metabolism like amino acids, nucleosides and fatty acids [
3]. Non-transformed cells predominantly use glycolysis and the TCA cycle to metabolize glucose to CO
2 and H
2O. Cancer cells on the other hand take up larger amounts of glucose compared normal cells and metabolize it through glycolysis at higher rates [
3]. The pyruvate produced by glycolysis in cancer cells is preferentially metabolized to lactate by cytoplasmic lactate dehydrogenases [
4]. This shift from ATP production through oxidative phosphorylation to ATP generation through glycolysis is called “aerobic glycolysis” or the “Warburg effect”. It occurs even in the presence of sufficient oxygen and was first described by Otto Warburg [
5].
Based on the prevalence of the enhanced glucose uptake and metabolism in cancer cells, positron emission tomography (PET) with the glucose analogue
18F-fluoro-2-deoxy-
d-glucose (FDG) is a non-invasive molecular imaging tool to detect many tumor entities and monitor therapeutic response by analysing tumor metabolic activity [
4,
6]. FDG, similar to glucose, is transported into the cell via glucose transporters (GLUTs) and is phosphorylated by hexokinase (HK) to FDG-6-phosphate. FDG-6-phosphate is trapped in the cell since it is no substrate for glycolytic or pentose phosphate pathways, unable to diffuse out of the cell and it is dephosphorylated slowly. Thus, FDG accumulates in the cell at a rate proportional to glucose utilization and tumors with an increased uptake of glucose can be visualized non-invasively by PET which also enables quantitative assessment of glucose utilization [
7,
8].
Metabolic changes in cancer were considered as a secondary effect of the transformation process. Molecular mechanisms that lead to metabolic reprogramming and to the metabolic phenotype of cancer cells are only partly understood and include activation of oncogenes and kinases like c-MYC, members of the phosphoinositide 3-kinase (PI3K) signalling pathway [PI3K, protein kinase B (AKT), mammalian target of rapamycin (mTOR)], stabilization of transcription factors like hypoxia-inducible factor 1 (HIF-1) and inactivation of tumor suppressor genes like p53 [
3,
4,
9]. Concordant with the increased uptake of glucose, expressions of GLUT genes, namely GLUT1 and genes of glycolytic enzymes are increased in many cancers [Review:
10]. Besides transcription factors, gene expression is regulated also by microRNAs (miRNAs) which are endogenous small non-coding RNAs of 18–25 nucleotides and regulate gene expression by binding to the 3′-untranslated region of the target mRNA [
11]. Depending on their target genes, miRNA can function as tumor-suppressors or oncogenic miRNAs. A few specific miRNAs are described to be involved in glucose metabolism in cancer cells including miR21, -23a,- 133a, -133b, -138-1 and -143 [
12‐
14].
Non-Hodgkin’s lymphoma (NHL) is a heterogeneous group of diseases and includes several lymphoma subtypes like diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma, follicular lymphoma and others [
15]. NHLs display enhanced FDG uptake and thus PET is used for staging, monitoring of therapy response and prognostication of these patients. High glucose uptake is seen as a surrogate marker of an aggressive tumor and associated with poor outcome in DLBCL [
16,
17]. Patients with NHL are usually treated with a chemotherapeutic regime including rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). However, only in 40–50% of patients a complete response is achieved after first therapy [
18]. Interim PET after a few cycles of chemotherapy may be used according to current guidelines for NHL patients to predict response but currently without a recommendation to change therapy in non-responders [
19,
20]. Clinical trials concerning the value of a change of the therapeutic regime in non-responding patients based on interim PET, e.g. the PETAL trial (PET-guided therapy of aggressive non-Hodgkin lymphoma) [
21,
22] are performed.
The involvement of PI3K, c-MYC and MAPK pathways on glucose uptake, expression of glycolysis-associated genes and cell proliferation in lymphoma is not known yet.
Thus in this study we performed experiments on the regulation of glucose uptake, transcription of genes involved in glycolysis, and regulation of miRNAs by the PI3K/AKT/mTOR pathway, c-MYC and different MAPK signaling pathways. Moreover, we studied the effect of 2-deoxy-d-glucose (2-DG), a glucose analogue that decreases glycolytic rate by inhibiting HK, as a single agent and in combination with inhibitors of the c-MYC-, PI3K-, and MAPK signaling pathway inhibitors on cell viability.
Discussion
In this study we showed regulation of glucose uptake by c-MYC- and PI3K/AKT/mTOR dependent pathways in lymphoma cell lines and demonstrated evidence for the altered glucose metabolism as a potential target to improve inhibitor-based therapeutic approaches in these cells.
During tumorigenesis, a change in energy metabolism of the tumor cells occurs and deregulated metabolism of glucose is one hallmark of cancer [
31]. The exact molecular mechanisms leading to this altered phenotype of cancer cells remain unclear. Besides hypoxia, activation of oncogenes and inactivation of tumor suppressor genes, alterations in the cellular signaling network are involved in the glycolytic switch in cancer cells [Review:
32].
Effects of PI3K/mTOR inhibitors
The PI3K/AKT/mTOR pathway is altered in many human cancers by activating mutations, aberrant receptor tyrosine kinase signaling or inactivating mutations in tumor suppressor genes like PTEN (phosphatase and tensin homolog) [
33,
34]. In NHL, this pathway is often activated, although mutations are only infrequently found [Review:
35]. AKT as a downstream effector of PI3K is known as an important driver of the tumor glycolytic phenotype and renders cancer cells dependent on glycolysis for survival [
9,
36]. AKT stimulates glucose uptake and glycolysis by increasing the expression and membrane translocation of glucose transporter proteins like GLUT1 and by activating glycolytic enzymes and regulating HK expression, activity and interaction with mitochondria [
37]. mTOR is a serine/threonine kinase downstream of PI3K-AKT that acts through the mTOR complexes 1 and 2 (mTORC1 and -2) to induce transcription of many genes involved in altered tumor cell metabolism [
38].
Treatment of our NHL cell lines with the PI3K inhibitor LY294002 or the mTOR inhibitor Rapamycin led to a decrease in cell viability in all three cell lines with IC50 values in the range of around 1 µM for Rapamycin and 10 µM for LY294002 (Fig.
4; Table
1) demonstrating the importance of the PI3K pathway for survival of the cells. These findings are in good agreement with recently reported results of Ezell and coworkers [
39] who found PI3K and mTOR inhibitors effective in arresting proliferation in DLBCL lines. However, response rates to mTOR inhibitors as single agent therapy in phase II clinical studies are only around 30% in NHL [
40,
41].
In addition to the effects on the number of viable cells, both inhibitors led to a decreased FDG uptake after 24 h treatment as expected (Fig.
3). A similar inhibitory effect of PI3K/mTOR inhibitors on FDG uptake was already described in cervical cancer cells [
42] and confirms the well-known importance of the PI3K pathway for enhanced glucose metabolism.
Unexpectedly, while Rapamycin led to a decrease in GLUT1 mRNA expression, treatment with LY294002 did not significantly affect GLUT1 mRNA in all three cell lines (Table
2). This discrepancy between mRNA results and FDG uptake after LY294002 treatment may be due to modifications in protein expression and processing like reduced translocation of GLUT1 and GLUT4 to the cell membrane as described in cervical cancer and lung adenocarcinoma cells [
42,
43]. In cutaneous T-cell lymphoma cells, incubation with Rapamycin decreased glucose uptake in both cell lines investigated, but mRNA expression of glycolysis genes was diminished only in one cell line [
44]. These results show that the molecular mechanism by which the PI3K signaling cascade regulates GLUT processing is still not clear and further experiments are needed to elucidate the modulation of expression, translocation and regulation of GLUTs.
The decrease of LDHA mRNA expression as well as the increase in G6Ptase after incubation with LY294002 and Rapamycin in all three cell lines corresponds to a diminished glycolysis after PI3K/AKT/mTOR inhibition, since LDHA is a glycolytic enzyme while G6Ptase catalyzes the reaction from glucose-6-phosphate back to glucose [
3].
HK2 mRNA expression was diminished in all three cell lines after incubation with Rapamycin, but not with LY294002. This may reflect the distinct levels of inhibition by the two substances. LY294002 inhibits PI3K, while Rapamycin inhibits mTOR proteins that are located downstream of PI3K and AKT. Treatment with LY294002 may therefore be circumvented by activation of the downstream AKT and mTOR kinases by alternative pathways or mutations and thus may explain the different effects of LY294002 and Rapamycin in our cells [
45].
The changes in miRNA expression after treatment with LY294002 or Rapamycin primarily reflect the oncogenic features of the PI3K/mTOR pathway in NHL cells with downregulation of the oncogenic miRNA21 and upregulation of the tumor suppressor miRNAs 133a, -133b and -138-1 (Table
3). Further experiments will show if this regulation is directly related to the diminished expression of the GLUT1 and LDHA genes after inhibition of this pathway and if manipulation of the miRNA level may influence expression of glycolysis-related genes.
Effect of the c-MYC inhibitor
c-MYC is an oncogenic transcription factor that is overexpressed in many cancer types including B- and T-cell malignancies and is involved in cell metabolism and cell proliferation [
46]. In lymphoma, c-MYC activation occurs by several molecular mechanisms including translocations, amplification, mutations, altered intracellular localization of the c-MYC protein or miRNA-dependent mechanisms [
47‐
49].
Incubation of our cells with the c-MYC inhibitor as expected led to a decreased number of viable cells (Fig.
4a) with IC50 values (6.48–10.23 µM) in the lower range seen in other cell systems (breast cancer cells: 20–30 µM [
50]; multiple myeloma cell lines: 12–45 µM [
51]; ovarian carcinoma cell lines: 3.2 and 4.4 µM [
52]; HepG2 hepatocellular carcinoma cells: around 100 µM [
53]; acute myeloid leukemia (AML) cell lines: 60–90 µM [
54]). Thus, the c-MYC inhibitor may be a suitable substance for reducing the number of viable lymphoma cells. Further experiments may reveal the mode of action, e.g. apoptosis induction, inhibition of proliferation and cell cycle arrest as well as its in vivo efficacy in lymphoma.
Besides a reduction of the number of viable cells, treatment of lymphoma cells with the c-MYC inhibitor led to a marked decrease in FDG uptake in all three cell lines with the SU-DHL-6 cells showing the most pronounced effect (Fig.
3). Corresponding to the decreased FDG uptake, after incubation with the c-MYC inhibitor the expression of GLUT1 mRNA was significantly decreased in all three cell lines (Table
2). Similar effects of a decreased glucose uptake and a diminished expression of GLUT 1 after incubation with the c-MYC inhibitor or after knockdown of c-MYC with siRNA were already described in breast cancer cells [
50]. Further experiments are necessary to evaluate protein expression and cellular localization of glucose transporters to determine the cellular effect of an inhibition of c-MYC.
As expected, expression of the mRNA encoding LDHA was also diminished after incubation with the c-MYC-inhibitor (Table
2) while expression of HK2 and the G6Pase was enhanced. Targeting of the expression of glycolytic enzymes by c-MYC was already described in other cell systems [
55,
56]. However, while we still do not have an explanation for the increased expression of HK2, enhanced G6Pase expression may be explained similarly as the one after inhibition of the PI3K pathway and may reflect a partly reversed Warburg effect.
The changes in miRNA expression after c-MYC inhibition resemble those after inhibition of PI3K/mTOR probably reflecting the function of c-MYC as an oncogene in NHL cells. In contrast to the results obtained after mTOR inhibition with Rapamycin, after treatment with the c-MYC inhibitor or LY294002, the expression of the miRNA133a was diminished (Table
3). Thus, c-MYC is involved in pathways stimulating proliferation and also enhances the glycolytic phenotype of lymphoma cells. Inhibition of c-MYC therefore is an attractive strategy to inhibit both the altered glucose metabolism and proliferation in lymphoma cells. Further experiments are needed to characterize the exact molecular relationship between glycolytic inhibition and cell death and its mechanisms.
Effect of inhibitors of MAPK pathways
The p38-MAPK signaling pathway is involved in many cellular functions like differentiation, proliferation and induction of cell death. The exact role of p38-MAPK in a cancer cell depends on the cell type and the tumor stage [
57]. In NHL, upregulation was shown in DLBCL [
58] and an increased level of phosphorylated p38-MAPK has been correlated with malignancy and failure of response to CHOP treatment [
59,
60]. In our experiments, we did not find an influence of p38-MAPK inhibition on cell survival and proliferation, FDG uptake and expression of glycolysis-related genes and miRNAs. These results fit those published by Vega et al. [
61] who reported on the lack of apoptosis-inducing effects of SB203580 in NHL cells. Furthermore, Elenitoba-Johnson et al. [
58] reported that SB203580 had no effect on proliferation in one of three NHL cell lines, while p38-MAPK inhibition was effective in the other two indicating that the effect of a p38-MAPK inhibition is dependent on presently unknown cell line characteristics.
The MEK pathways are activated mainly in response to the stimulation of tyrosine kinase receptors [
62]. MEK pathways are shown to be involved in an enhanced glucose uptake and the metabolic shift in cancer types with an activated BRAF-MAPK pathway by BRAF mutations like those found in melanoma [
63]. Furthermore, MAPK pathways were shown to interact with enhanced glutamine metabolism in melanoma cells: In BRAF-mutated melanoma cells that were MAPK inhibitor-resistant, a greater uptake of glutamine and an increased sensitivity to glutamine was demonstrated compared to MAPK inhibitor-sensitive cells [
64]. In addition, inhibitors of glutaminase were more efficient in MAPK-inhibitor-resistant cells with regard to decreased cell survival indicating that besides glucose metabolism, glutamine metabolism may be a suitable therapeutic target in cancer cells [
64]. In lymphoma, no mutational activation of members of the MAPK pathways are described except in pediatric-type nodal follicular lymphoma, a variant of follicular lymphoma with invariably benign behavior, with a mutation frequency in the
MEK1 gene of 43% [
65]. The data presented here show no influence of the MEK inhibitor PD98059 on the number of viable cells, glucose uptake and the expression of glycolysis-related genes and thus fit these literature data. Interestingly, we found an upregulation of the tumor suppressor miRNAs133a, -133b and -138-1 after PD98059 treatment indicating the involvement of MAPK in presently unknown oncogenic pathways.
Effect of the glucose analog 2-DG
The glucose analog 2-DG is an inhibitor of HK that is used to block the Warburg effect in cancer cells [
66]. 2-DG is taken up into the cells via GLUTs and phosphorylated by HK to 2-deoxyglucose-6-phosphate which cannot be further metabolized and thus accumulates in the cell and interferes with the glycolytic pathway by inhibiting HK and phosphoglucose isomerase [
66,
67].
Due to its ability to inhibit glycolysis, 2-DG has been evaluated as an anticancer agent in several cell systems [Review:
68]. In our lymphoma cell lines we found a decrease in cell viability after incubation with 2-DG for 48 h with IC50 values in the range of 2.86 mM to 4.65 mM with SU-DHL-6 being the most sensitive cell line (Fig.
4f; Table
1). The IC50 values found here are in the lower range of values reported in the literature for other cell systems (MCF7 breast cancer cells: 6.7 mM; LNCaP prostate cancer cells: 8.1 mM [
69]).
Although 2-DG decreases the number of viable cells in short-time cell culture experiments, it has not been effective as a single agent in vivo [
68]. We therefore combined 2-DG with the inhibitors used in this study and investigated the effects of combined inhibition on cell viability (Table
2). Approx. half the concentrations used in the other experiments were used for combined treatment (Table
2). Synergistic effects were observed with the c-MYC-inhibitor 10058-F4, with LY294002 and with the p38 MAPK inhibitor SB203580 as well as with Rapamycin in 2 of 3 cell lines (Table
2).
A synergistic effect of a combined treatment with 2-DG and PI3K/mTOR inhibitors as found in our experiments has already been described by a few authors: In lung cancer cell lines, an analogue of Rapamycin hypersensitized cells to 2-DG treatment under hypoxic conditions [
70]. Furthermore, a dual PI3K/mTOR inhibitor has recently been reported to have synergistic effect with 2-DG on cell survival in two cell lines of primary effusion lymphoma (PEL), a rare subtype of B-cell NHL [
71]. A possible explanation for the synergistic action of inhibitors of the PI3K/mTOR pathways with inhibitors of glycolysis was recently found in cells derived from various cancer types [
72]. These authors reported on an escape from glycolysis addiction of tumor cells by an mTORC1-dependent circumvention of the 2-DG-mediated glycolysis block via the pentose phosphate pathway back to glycolysis [
72].
Combined treatment with 2-DG and SB203580 has recently been described in pancreatic and ovarian cell lines [
73] with similar synergistic effects on cell survival in five of six cell lines as described here. On the other hand, Cheng et al. reported that the p38-MAPK pathway is necessary for apoptosis induced by 2-DG in pancreatic cancer cells [
74]. Taken together, these data suggest that the effect of p38-MAPK inhibitors depends on the individual cell context and on activation pattern of signaling pathways within the cell.
Up to now, to the best of our knowledge, no data on the efficacy of a combined treatment with 2-DG and the c-MYC inhibitor or MEK inhibitor like PD98059 are available. Inhibition of c-MYC alone resulted in significant decrease of viable cells which was synergistically enhanced by 2-DG in our cells (Table
3). On the other hand, combination of the MEK inhibitor PD98059 with 2-DG showed a better effect than treatment with 2-DG alone (Table
3). Although we do not know the molecular reasons at this time, combined treatment of NHL cells with 2-DG and inhibitors of the PI3K/AKT pathway, c-MYC and p38 MAPK intracellular signaling pathways may be a promising new therapeutic option. Further experiments will provide further insights into the molecular background of cell inhibition and the mechanism of cell death induced by these substances.
Authors’ contributions
MBP drafted the project design, planned and conducted experiments, analyzed the data and wrote the text. NBB conducted experiments and analysed data. AB contributed to project design and discussion. UD participated in project design, writing and discussion. SM contributed to project design, FDG uptake assay, text writing and discussion of the data and text. All authors read and approved the final manuscript.