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Review
. 2014 Oct 14;20(38):13705-17.
doi: 10.3748/wjg.v20.i38.13705.

Mechanisms of regulation of PFKFB expression in pancreatic and gastric cancer cells

Oleksandr H Minchenko  1 , Katsuya Tsuchihara  1 , Dmytro O Minchenko  1 , Andreas Bikfalvi  1 , Hiroyasu Esumi  1
Affiliations
Review

Mechanisms of regulation of PFKFB expression in pancreatic and gastric cancer cells

Oleksandr H Minchenko et al. World J Gastroenterol. .

Abstract

Enzymes 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and -4 (PFKFB-3 and PFKFB-4) play a significant role in the regulation of glycolysis in cancer cells as well as its proliferation and survival. The expression of these mRNAs is increased in malignant tumors and strongly induced in different cancer cell lines by hypoxia inducible factor (HIF) through active HIF binding sites in promoter region of PFKFB-4 and PFKFB-3 genes. Moreover, the expression and hypoxia responsibility of PFKFB-4 and PFKFB-3 was also shown for pancreatic (Panc1, PSN-1, and MIA PaCa-2) as well as gastric (MKN45 and NUGC3) cancer cells. At the same time, their basal expression level and hypoxia responsiveness vary in the different cells studied: the highest level of PFKFB-4 protein expression was found in NUGC3 gastric cancer cell line and lowest in Panc1 cells, with a stronger response to hypoxia in the pancreatic cancer cell line. Overexpression of different PFKFB in pancreatic and gastric cancer cells under hypoxic condition is correlated with enhanced expression of vascular endothelial growth factor (VEGF) and Glut1 mRNA as well as with increased level of HIF-1α protein. Increased expression of different PFKFB genes was also demonstrated in gastric, lung, breast, and colon cancers as compared to corresponding non-malignant tissue counterparts from the same patients, being more robust in the breast and lung tumors. Moreover, induction of PFKFB-4 mRNA expression in the breast and lung cancers is stronger than PFKFB-3 mRNA. The levels of both PFKFB-4 and PFKFB-3 proteins in non-malignant gastric and colon tissues were more pronounced than in the non-malignant breast and lung tissues. It is interesting to note that Panc1 and PSN-1 cells transfected with dominant/negative PFKFB-3 (dnPFKFB-3) showed a lower level of endogenous PFKFB-3, PFKFB-4, and VEGF mRNA expressions as well as a decreased proliferation rate of these cells. Moreover, a similar effect had dnPFKFB-4. In conclusion, there is strong evidence that PFKFB-4 and PFKFB-3 isoenzymes are induced under hypoxia in pancreatic and other cancer cell lines, are overexpressed in gastric, colon, lung, and breast malignant tumors and undergo changes in their metabolism that contribute to the proliferation and survival of cancer cells. Thus, targeting these PFKFB may therefore present new therapeutic opportunities.

Keywords: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3; 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4; Gastric cancer; Hypoxia; Hypoxia inducible factor; Lung cancer; MKN45; NUGC3; PST-1; Panc1.

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Figures

Figure 1
Figure 1
Effect of hypoxia (H) and hypoxia mimic dimethyloxalylglycine (I) on the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and -4 mRNA in human gastric cancer cell lines MKN45 and NUGC3 and pancreatic cancer cell line Panc1. Measured by ribonuclease protection assay, N: Control (normoxic) cells[32]. PFKFB: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Figure 2
Figure 2
Quantification of ribonuclease protection assay of the effect of hypoxia (H) on the expression level of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4 and -3 mRNAs in human gastric (MKN45 and NUGC3) and pancreatic (Panc1) cancer cell lines. bP < 0.01 vs control cells; dP < 0.01 vs control cells[32]. N: Normoxic (control) cells. PFKFB: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Figure 3
Figure 3
Western blot analysis of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4 protein in human gastric (MKN45 and NUGC3) and pancreatic (Panc1) cancer cell lines: Effect of hypoxia (H) and dimethyloxalylglycine (I)[32]. PFKFB: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Figure 4
Figure 4
Expression of hypoxia inducible factor-1α protein (Western blotting; A) and hypoxia inducible factor-1α and hypoxia inducible factor-2α mRNA (ribonuclease protection assay; B) in human gastric (MKN45 and NUGC3) and pancreatic (Panc1) cancer cell lines: effect of hypoxia (H) and dimethyloxalylglycine (I)[32]. HIF: Hypoxia inducible factor.
Figure 5
Figure 5
Effect of hypoxia (H) and hypoxia mimic dimethyloxalylglycine (I) on the expression level of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3, -4, and GLUT1 mRNAs (measured by qPCR) in human pancreatic (PSN-1) cancer cells. n = 4; bP < 0.01 vs control cells. N: Normoxic (control) cells; PFKFB: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Figure 6
Figure 6
Effect of hypoxia (H) and hypoxia mimic dimethyloxalylglycine (I) on the expression level of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3, -4, and GLUT1 mRNAs (measured by qPCR) in human pancreatic (MIA PaCa-2) cancer cells. n = 4, bP < 0.01 vs control cells. N: Normoxic (control) cells; PFKFB: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Figure 7
Figure 7
Representative polyacrylamide gel autoradiograph employed in a typical ribonuclease protection assay of different 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase genes (PFKFB-1, PFKFB-2, PFKFB-3, and PFKFB-4), GLUT1, hypoxia inducible factor-1α, and different alternative splice variants of VEGF-A in gastric malignant tumors (T) and non-malignant tissue counterparts (N) from same patients. The 18S rRNA expressions were used as control of RNA quantity used for analysis[32]. HIF: Hypoxia inducible factor; PFKFB: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Figure 8
Figure 8
Quantification of ribonuclease protection assay of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-1, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4, GLUT1, hypoxia inducible factor-1α, and splice variants of VEGF-A mRNA in human gastric malignant tumors (T) and corresponding non-malignant tissue (N) from the same patients. bP < 0.01 vs control cells[32]. HIF: Hypoxia inducible factor; PFKFB: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; VEGF: Vascular endothelial growth factor.
Figure 9
Figure 9
Quantification of ribonuclease protection assay of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4 mRNA expressions in lung, colon, and breast malignant tumors (T) and corresponding non-malignant tissue counterparts (C). Values of PFKFB-3 and PFKFB-4 mRNA expressions were normalized to 18S rRNA; n = 15-20, bP < 0.01 vs non-malignant tissues[30,33]. PFKFB: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Figure 10
Figure 10
Quantification of ribonuclease protection assay of VEGF and Glut1 mRNA expressions in lung, colon, and breast malignant tumors (T) and corresponding non-malignant tissue counterparts (C). Values of VEGF and Glut1 mRNA expressions were normalized to 18S rRNA; n = 15-20; bP < 0.01 vs non-malignant tissues[30,33]. PFKFB: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; VEGF: Vascular endothelial growth factor.
Figure 11
Figure 11
Representative Western blot analysis of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3, inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4, and hypoxia inducible factor-1α protein levels in breast, lung, colon, and stomach malignant tumors (T) and non-malignant (control) tissues counterparts (N) from same patients. The actin was used to ensure equal loading of the sample[30,32,33]. HIF: Hypoxia inducible factor; PFKFB: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Figure 12
Figure 12
Representative polyacrylamide gel autoradiograph employed in a typical ribonuclease protection assay of endogenous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 mRNA in pancreatic carcinoma cell line Panc1, stable transfected by pcDNA3.1(+) vector (Panc1 cells) or by dominant/negative 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (Panc1 + dnPFKFB-3) in normoxic (N) condition and after treatment of Panc1 cells with dimethyloxalylglycine, inhibitor of prolyl hydroxylase (I; 1 mmol/L for 6 h). The 18S rRNA antisense probe was used as control of analyzed RNA quantity[89]. PFKFB: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Figure 13
Figure 13
Quantification of ribonuclease protection assay of endogenous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 mRNA expression in pancreatic carcinoma cell line Panc1, stable transfected by pcDNA3.1(+) vector or dominant/negative 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 in normoxic (control) condition (C) and after treatment of Panc1 cells with dimethyloxalylglycine, inhibitor of prolyl hydroxylase (I). n = 5; bP < 0.01 vs control[89]. dnPFKFB-3: Dominant/negative 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3.
Figure 14
Figure 14
Endogenous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4, and vascular endothelial growth factor mRNA expressions in pancreatic carcinoma cell line PSN-1, stable transfected with pcDNA3.1(+) vector (Vector), dominant/negative 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3, and dominant/negative 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4, measured by ribonuclease protection assay. n = 5; aP < 0.05 vs control; bP < 0.01 vs control[89]. PFKFB: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; VEGF: Vascular endothelial growth factor.
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