Phosphorylation of pyruvate kinase M2 and lactate dehydrogenase A by fibroblast growth factor receptor 1 in benign and malignant thyroid tissue

Abstract

Background: Lactate dehydrogenase A (LDHA) and Pyruvate Kinase M2 (PKM2) are important enzymes of glycolysis. Both of them can be phosphorylated and therefore regulated by Fibroblast growth factor receptor 1 (FGFR1). While phosphorylation of LDHA at tyrosine10 leads to tetramerization and activation, phosphorylation of PKM2 at tyrosine105 promotes dimerization and inactivation. Dimeric PKM2 is found in the nucleus and regulates gene transcription. Up-regulation and phosphorylation of LDHA and PKM2 contribute to faster proliferation under hypoxic conditions and promote the Warburg effect.

Methods: Using western blot and SYBR Green Real time PCR we investigated 77 thyroid tissues including 19 goiter tissues, 11 follicular adenomas, 16 follicular carcinomas, 15 papillary thyroid carcinomas, and 16 undifferentiated thyroid carcinomas for total expression of PKM2, LDHA and FGFR1. Additionally, phosphorylation status of PKM2 and LDHA was analysed. Inhibition of FGFR was performed on FTC133 cells with SU-5402 and Dovitinib.

Results: All examined thyroid cancer subtypes overexpressed PKM2 as compared to goiter. LDHA was overexpressed in follicular and papillary thyroid cancer as compared to goiter. Elevated phosphorylation of LDHA and PKM2 was detectable in all analysed cancer subtypes. The highest relative phosphorylation levels of PKM2 and LDHA compared to overall expression were found in undifferentiated thyroid cancer. Inhibition of FGFR led to significantly decreased phosphorylation levels of PKM2 and LDHA.

Conclusions: Our data shows that overexpression and increased phosphorylation of PKM2 and LHDA is a common finding in thyroid malignancies. Phospho-PKM2 and Phospho-LDHA could be valuable tumour markers for thyroglobulin negative thyroid cancer.

Figure 1

Protein analysis of PKM2, LDHA and FGFR1 expression in thyroid tissues. Representative images of western blot performed with total protein lysates obtained from thyroid tissues and antibodies against total-PKM2, Phospho-PKM2, total-LDHA, Phospho-LDHA and FGFR1. Thyroid samples were divided into following histological subgroups: benign goiter (Goiter), follicular adenoma (FA), follicular thyroid cancer (FTC), papillary thyroid cancer (PTC) and undifferentiated thyroid cancer (UTC). ß-actin was used as a normalizing marker.

Figure 2

Evaluation of total- and Phospho-PKM2 protein expression. Total and y105-phosphorylated PKM2 expressions in goiter, FA, FTC, PTC and UTC were analysed by employment of western blot analysis and evaluated densitometrically with ImageJ software. Protein expression was measured in relation to FTC133 as positive control and then normalized to the expression of ß-actin as normalizing marker. Differences are expressed as percent to positive control defined as 100%. *p < 0.05 indicates a statistical significance.

Figure 3

Phospho-PKM2/total PKM2 ratio. Total and y105-phosphorylated PKM2 expression in goiter, FA, FTC, PTC and UTC were analysed by employment of western blot (see Figure 1). Phospho-PKM2/total-PKM2 ratio was built to show phospho-PKM2 in relation to total-PKM2. *p < 0.05 indicates a statistical significance.

Figure 4

Correlations between Phospho-PKM2/total-PKM2. Correlation between ratio of Phospho-PKM2/total-PKM2 and FGFR1 expression. Pearson correlation was applied.

Figure 5

Correlations between Phospho-LDHA/total-LDHA and FGFR1 expression. Correlation between ratio of LDHA/total-LDHA and FGFR1 expression. Pearson correlation was applied.

Figure 6

mRNA expression of Pyruvate Kinase M1 and M2 (PKM1/2). Expression of PKM1/2 in goiter, FA, FTC, PTC and UTC was analysed by employment of Q-RT-PCR. mRNA expression was measured in relation to FTC133 as positive control and then normalized to the expression of yWHAZ and GAPDH as normalizing markers. Differences are expressed as percent to positive control defined as 100%. *p < 0.05 indicates a statistical significance.

Figure 7

Evaluation of total- and Phospho-LDHA protein expression. Total and y10-phosphorylated LDHA expressions in goiter, FA, FTC, PTC and UTC were analysed by employment of western blot analysis and evaluated densitometrically with ImageJ software. Protein expression was measured in relation to FTC133 as positive control and then normalized to the expression of ß-actin as normalizing marker. Differences are expressed as percent to positive control defined as 100%. *p < 0.05 indicates a statistical significance.

Figure 8

Phospho-LDHA / total-LDHA ratio. Total and y10-phosphorylated LDHA expression in goiter, FA, FTC, PTC and UTC were analysed by employment of western blot (see Figure 1). Phospho-PKM2 / total-PKM2 ratio was built to show phosphorylated PKM2 in relation to total PKM2. *p < 0.05 indicates a statistical significance.

Figure 9

mRNA expression of Lactate dehydrogenase A (LDHA). Expression of LDHA in goiter, FA, FTC, PTC and UTC was analysed by employment of Q-RT-PCR analysis. mRNA expression was measured in relation to FTC133 as positive control and then normalized to the expression of yWHAZ and GAPDH as normalizing markers. Differences are expressed as percent to positive control defined as 100%. *p < 0.05 indicates a statistical significance.

Figure 10

mRNA expression of Fibroblast growth factor receptor 1 (FGFR1). Expression of FGFR1 in goiter, FA, FTC, PTC and UTC was analysed by employment of Q-RT-PCR analysis. mRNA expression was measured in relation to FTC133 as positive control and then normalized to the expression of yWHAZ and GAPDH as normalizing markers. Differences are expressed as percent to positive control defined as 100%. *p < 0.05 indicates a statistical significance.

Figure 11

Evaluation of FGFR1 protein expression. Expression of FGFR1 in goiter, FA, FTC, PTC and UTC was analysed by employment of western blot analysis and evaluated densitometrically with ImageJ software. Protein expression was measured in relation to FTC133 as positive control and then normalized to the expression of ß-actin as normalizing marker. Differences are expressed in percent to positive control defined as 100%. *p < 0.05 indicates a statistical significance.

Figure 12

Inhibition of FGFR and response in phosphorylation status of PKM2 and LDHA. Inhibiton experiments were performed with Dovitinib and SU-5402, two inhibitors of Fibroblast growth factor receptor (FGFR). Phosphorylation status of PKM2 and LDHA was measured after four hours and showed a significant decrease with Dovitinib 100nM and SU-5402 20 μM for both proteins. DMSO was used as positive control. No significant downregulation of PKM2 and LDHA phosphorylation was detectable with 1nM and 10 nM of Dovitinib.