Background
The Warburg effect describes a general feature of cancer cells to show elevated glucose uptake and lactate production even in the presence of oxygen [
1]. Warburg proposed an impaired glucose oxidation, which leads to extensive excretion of lactate under normoxia [
2]. However, recent data showed that the Warburg effect is common not only in cancer cells but also in induced pluripotent stem cells [
3] and in proliferating T cells [
4]. These findings raise many questions related to cancer specific alterations in glycolysis and their possible use as prognostic or therapeutic targets.
Pyruvate Kinase (PK), catalysing the step from phosphoenolpyruvate to pyruvate, is a key enzyme of glycolysis. Furthermore it is an important regulator of the Warburg effect. In thyroid tissue there are two isoenzymes: pyruvate kinase M1 (PKM1) and pyruvate kinase M2 (PKM2), which result from alternative splicing of the PKM gene [
5]. Bluemlein et al. showed that PKM2 is the dominant isoenzyme in all examined benign and malignant thyroid tissues [
6] (Additional file
1). Higher levels of PKM2 in tumour tissues contribute to growth advantage and faster progression in xenograft models as compared to cancer cells expressing PKM1 [
7]. However, elevated levels of inactivated dimeric PKM2 are found in cancer cells [
8]. This inactivation may be promoted by different mechanisms [
9,
10] and suggests that PKM2 may possess other, non-glycolytic functions such as regulation of transcription. In addition to these effects involvement of other proteins, which may dramatically affect the function of PKM2, has been reported [
10,
11]. It has been demonstrated that phosphorylation of tyrosine 105 of PMK2 by fibroblast growth factor receptor 1 (FGFR1) prevents tetramerization and inactivates PKM2. As a consequence this leads to faster proliferation under hypoxic conditions and increased tumour growth in xenograft models [
12]. The enzymatically inactivated dimeric form of PKM2 can be translocated to the nucleus and may act as a protein kinase regulating gene transcription implicated in tumour growth [
13-
16]. Inactivation of PKM2 leads to accumulation of upstream glycolytic metabolites and activation of the pentose-phosphate pathway, hexosamine-pathway and serine biosynthesis. This results in increased availability of metabolites for redox control and nucleotide biosynthesis [
11,
17]. With regard to clinical employment as a tumourmarker, a great diagnostic and prognostic potential of PKM2 has been demonstrated for several malignancies including oesophagus, pancreas or colorectal cancer [
18,
19]. However, data concerning PKM2 in thyroid cancer is still lacking.
Lactate dehydrogenase (LDH) catalyses the conversion from pyruvate to lactate. Active LDH consists of four monomers. The two different monomers lactate dehydrogenase A (LDHA) and lactate dehydrogenase B (LDHB) are expressed in an organ depending manner. While LDHA preferentially turns pyruvate into lactate, LDHB works the opposite way [
20].
LDHA is up-regulated in a wide range of tumour tissues including lung, breast, endometrium, urinary bladder, testicular germ cell and large intestine cancers [
20,
21]. Down-regulation or inhibition of LDHA resulted in decreased ATP levels, reduced mitochondrial membrane potential and an increase in oxidative stress that is linked to cell death. Furthermore, decreased levels of LDHA were related to inhibition of tumour xenograft maintenance and progression [
22,
23]. Investigations on thyroid tissues showed a decreased LDHA/LDHB ratio in thyroid oncocytoma and follicular tumours compared to normal thyroid tissue which was altered by estrogen related receptor alpha. Those tumours were more dependent on oxidative phosphorylation [
24]. In contrast to PKM2, FGFR1-mediated phosphorylation of LDHA at tyrosine 10 (y10) promotes tetramerization and turns LDHA into the active enzyme [
25].
Fibroblast growth factor receptors (FGFR) play an important role in many human malignancies, such as bladder or breast cancer [
26,
27]. They are involved in the regulation of cellular proliferation, differentiation, migration and cell survival [
28]. With regard to thyroid tissue, the most studied receptor of this family, FGFR1, is overexpressed in differentiated thyroid cancer and in thyroid hyperplasia [
29,
30].
So far, FGFR1-mediated phosphorylation effects on LDHA and PKM2 have only been demonstrated in cell culture experiments without any relevance to human cancer tissue, especially thyroid carcinoma.
In this study we investigated the expression of PKM2, LDHA and FGFR1 in thyroid benign and malignant tissues by employment of qPCR and western blot. Additionally, a possible impact of FGFR1-mediated phosphorylation of PKM2 and LDHA on thyroid malignancy was evaluated.
Methods
Tissue
A total of 77 thyroid tissue samples, including 19 goiter tissues, 11 follicular adenomas (FA), 16 follicular carcinomas (FTC), 15 papillary thyroid carcinomas (PTC) and 16 undifferentiated thyroid carcinomas (UTC) were collected from patients at the surgical institute of the Martin Luther University Halle Wittenberg. The study was approved by the ethical committee of the medical department of the Martin Luther University Halle Wittenberg. All patients gave written consent.
Protein isolation and western blot analysis
Frozen tissue samples were collected directly after surgery and stored in liquid nitrogen. Tissue specimens were divided and sections where prepared and stained with hematoxylin and eosin to confirm the pathological diagnosis. After confirmation frozen tissue specimens were homogenised and further divided for RNA- or protein extraction. For protein extraction the homogenate was lysed using a protein lysis buffer (7 M urea, 2 M thiourea, 4% Chaps, 40 mM DTT). Protein concentration was measured by using the Bradford assay method. Proteins were separated by SDS-PAGE blotted on PVDF membrane and blocked with 5% BSA for one hour. They were incubated overnight in a 1:1000 dilution of the primary antibody. β-actin was used as normalizing marker. Total lysate from cell line FTC133 was used as positive control. FTC133 is a thyroid cancer cell line, which was obtained from a lymph node metastases from a 42-year-old male with follicular thyroid cancer. As proven in preliminary experiments FTC133 expresses PKM1, PKM2, LDHA and FGFR1 on mRNA and protein levels (Additional file
2). Membranes were first stained with Phospho-PKM2 or Phospho-LDHA antibody, respectively. Subsequently, they were treated with stripping buffer (15 g glycin, 1 g SDS, 10 ml Tween adjusted to pH 2.2 and filled up with H
2O to 1 l) and tested for remaining signals. They were again incubated overnight with total LDHA or PKM2 antibody, respectively. Protein expression was measured in relation to FTC133 as positive control and then normalized to the expression of ß-actin as a normalizing marker. Differences are expressed in percent to positive control defined as 100%. In order to evaluate the expression and phosphorylation status of PKM2 and LDHA we subjected total protein lysates to western blot with specific antibodies raised against total PKM2 and LDHA, phosphorylated y105 of PKM2 and phosphorylated y10 od LDHA. To evaluate the expression of FGFR1 antibodies against total FGFR1 were used (Table
1).
PKM2 Antibody | Cell Signaling Technology, Cambridge, England |
Phospho-PKM2 (Tyr105) Antibody | Cell Signaling Technology, Cambridge, England |
LDHA (C4B5) Rabbit mAb | Cell Signaling Technology, Cambridge, England |
Phospho-LDHA (Tyr10) Antibody #8176 | Cell Signaling Technology, Cambridge, England |
FGF Receptor 1 (D8E4) | Cell Signaling Technology, Cambridge, England |
Monoclonal Anti-ß-Aktin Clone AC15 | Sigma Aldrich, Saint Louis, USA |
Goat anti Rabbit IgG HRP | Santa Cruz Biotechnology, Santa Cruz, USA |
Goat anti Mouse IgG HRP | Santa Cruz Biotechnology, Santa Cruz, USA |
PKM1 Rabbit Polyclonal Ab | Signalway Antibody, Maryland, USA |
Q-RT-PCR
The transcripts of FGFR1, PKM and LDHA were investigated by employment of quantitative RT-PCR and primer pairs listed in Table
2. 1 μg of total RNA was reversely transcribed by using SuperScript II Kit according to the manufacturer’s instructions (Invitrogen). qPCR was conducted with Rotor Gene SYBR Green PCR Kit (Qiagen) on Rotor Gene Q® Qiagen 2 plax Platform (Qiagen). The cycling conditions were as follows: PCR initial activation step for 5 min. at 95°C, 40 cycles of two step cycling which included denaturation for 5 sec at 95°C and 10 sec of combined annealing and extension at the individual primer temperature between 57°C and 62.5°C. Relative mRNA levels of the transcripts were measured in relation to FTC133 as positive control and then normalized to the expression of the housekeeping genes YWHAZ and GAPDH. Differences are expressed in percent in relation to positive control defined as 100%.
PK-M1/2 | 5′CTG GGA AGC CTG TCA TCT GT-3′ | 5′- AGT CCC CTT TGG CTG TTT CT-3′ |
LDHA | 5′-GGC CTC TGC CAT CAG TAT CT-3′ | 5′-GCC GTG ATA ATG ACC AGC TT-3′ |
FGFR1 | 5′-ACA CTG CGC TGG TTG AAA A- 3′ | 5′-TGG TAT GTG TGG TTG ATG CTC- 3′ |
yWHAZ (Housekeeping) | 5′-AGC AGG CTC AGC GAT ATG AT-3′ | 5′-TCT CAG CAC CTT CCG TCT TT-3′ |
GAPDH (Housekeeping) | 5′-ACC CAG AAG ACT GTG GAT GG-3′ | 5′-TTC TAG ACG GCA GGT CAG GT-3′ |
Cell culture experiments
8505C and FTC133 cells were grown in DMEM/F12 suppplemented with 10% FCS and 1% PenStrep and incubated at 37°C, 5% CO2. For B-CPAP RPMI 1640 medium was used. FGFR1 inhibition experiments were performed on FTC133 cells by employment of Receptor Tyrosine Kinase Inhibitors TKI-258 (Dovitinib, Biomol) and SU-5402 (Sigma-Aldrich). Inhibition was conducted over 4 h with the indicated inhibitor concentrations. Control cells received corresponding concentrations of DMSO.
Statistical analysis
Statistical analyses were performed with IBM SPSS (version 20) software by employment of a priori test. In case of p < 0.05 a Kruskal Wallice and Mann Whitney U test for subgroup analysis was performed. For correlation analysis the Pearson coefficient was used. Boxplots were performed according to Tukey’s definition.
Discussion
In this study we demonstrated that total and phosphorylated PKM2 and LDHA proteins are significantly up-regulated in thyroid cancer tissues as compared to goiter. To our knowledge, in addition to previous PKM2 results in thyroid cancer [
6], this is the first report demonstrating an increased phosphorylation status of both proteins in thyroid cancer.
It is well known that in addition to the Warburg effect, expression of PKM2 and LDHA in tumour tissues may be regulated by Hypoxia inducible factor 1-alpha (HIF1a) and c-Myc. Both oncogenes were also reported to be elevated in thyroid malignancies [
31-
36]. With regards to our data, we found that in thyroid carcinoma tissues PKM2 is expressed more abundantly than LDHA. Based on these observations we suggest that the Warburg effect is not the only factor affecting PKM2 regulation in thyroid cancer, especially in UTC. Furthermore, as reported in our study, different expressional changes observed between PKM2 and LDHA may result from other than the enzymatic role of PKM2 in gene transcription and proliferation [
16,
37,
38].
Various other studies had previously demonstrated increased levels of LDHA and PKM2 in tumour tissues and their important role in proliferation and survival of malignant cells [
7,
23,
39]. In accordance with these findings we were able to show that thyroid carcinoma samples revealed not only significantly elevated levels of total PKM2 and LDHA compared to goiter or FA, but this expressional increase correlated directly with phosphorylated forms of both proteins. Furthermore, our data suggests that in patients with thyreoglobulin-negative thyroid cancer, phosphorylated PKM2 and LDHA may represent a more valuable diagnostic potential than total expressions of these proteins. Indeed, we found that the FGFR1 expression correlated well with phosphorylation status of both proteins. These results suggest a possible, but not only FGFR1-mediated phosphorylation mechanism of PKM2 and LDHA in thyroid carcinoma. However, increased phosphorylation of various proteins and up-regulation of FGFR are known to occur in cancer, being a possible confounder of our conclusion.
We therefore conducted additional cell culture experiments and were able to demonstrate that phosphorylation of PKM2 and LDHA occurs in an FGFR1-specific manner. Inhibition of FGFR1 in the thyroid cancer cell line FTC133 resulted in significantly decreased phosphorylation status of both investigated enzymes. It is worth noting that tyrosine kinase inhibitors like Dovitinib or SU-5402 could be a therapeutic option to target the Warburg effect in thyroid cancer cells.
Discrimination between follicular adenoma (FA) and FTC is often a challenge. Based on our data we noticed a significantly increased relative phosphorylation of PKM2 and LDHA (Figures
3 and
8) in FTC in comparison to FA. However, studies with early stage follicular thyroid cancer are necessary to determine whether relative phosphorylation could be a tool to discriminate FTC from FA.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
PK, CH, HD and BT designed the experiments. PK and HP performed the experiments. PK wrote the manuscript and BT revised the manuscript. PK and CS analysed the data. All authors read and approved the manuscript.