Hypoxia induces transcription of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 gene via hypoxia-inducible factor-1alpha activation.

The PFKFB4 gene encodes isoenzyme of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB or PFK-2/FBPase-2) which originally was found in the testes. We have studied hypoxic regulation of PFKFB4 gene in prostate cancer cell line, PC-3, and several other cancer cell lines. It was shown that hypoxia significantly induced PFKFB4 mRNA levels in PC-3 as well as in HeLa, Hep3B and HepG2 cell lines. Hypoxia increased PFKFB4 protein levels also. Moreover, desferrioxamine and cobalt chloride, which are known to mimic hypoxia, also had a stimulatory effect on the expression of PFKFB4 mRNA. In order to investigate the mechanisms of hypoxic regulation of PFKFB4 gene expression, we used dimethyloxalylglycine, which has the ability to mimic effect of hypoxia by significant induction of hypoxia-inducible factor (HIF-1alpha) protein levels. Our studies showed that PFKFB4 mRNA expression in PC-3, HeLa, Hep3B and HepG2 cell lines was highly responsive to dimethyloxalylglycine, an inhibitor of HIF-1alpha hydroxylase enzymes, suggesting that the hypoxia responsiveness of this gene is regulated by HIF proteins. To better understand the hypoxic regulation of PFKFB4 gene expression, we isolated genomic DNA, which includes the promoter region of PFKFB4. Cell transfection, deletion and site-specific mutagenesis of the PFKFB4 promoter region indicates that hypoxic induction of PFKFB4 gene expression is mediated by the hypoxia-responsive element (HRE). These experiments identified a HRE 422-429 bp upstream from the translation start site. Thus, our results indicate that testis-specific form of PFKFB or PFK-2/FBPase-2 is also expressed in several cancer cell lines and that hypoxia induces transcription of PFKFB4 gene in these cell lines by HIF-1alpha dependent mechanism. HRE in 5'-promoter region of PFKFB4 gene mediates hypoxic induction of PFKFB4 gene transcription.

Minchenko O ，Opentanova I ，Minchenko D ，Ogura T ，Esumi H ... -  《FEBS LETTERS》

Hypoxic regulation of PFKFB-3 and PFKFB-4 gene expression in gastric and pancreatic cancer cell lines and expression of PFKFB genes in gastric cancers.

Previously we have shown that hypoxia strongly induces the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and -4 (PFKFB-3 and PFKFB-4) genes in several cancer cell lines via a HIF-dependent mechanism. In this paper we studied the expression and hypoxic regulation of PFKFB-4 and PFKFB-3 mRNA as well as its correlation with HIF-1alpha, HIF-2alpha, VEGF and Glut1 mRNA expression in the pancreatic cancer cell line Panc1 and two gastric cancer cell lines MKN45 and NUGC3. This study clearly demonstrated that PFKFB-3 and PFKFB-4 mRNA are expresses in MKN45, NUGC3 and Panc1 cancers cells and that both genes are responsive to hypoxia in vitro. However, their basal level of expression and hypoxia responsiveness vary in the different cells studied. Particularly, PFKFB-3 mRNA is highly expressed in MKN45 and NUGC3 cancer cells, with the highest response to hypoxia in the NUGC3 cell line. The PFKFB-4 mRNA has a variable low basal level of expression in both gastric and pancreatic cancer cell lines. However, the highest hypoxia response of PFKFB-4 mRNA is found in the pancreatic cancer cell line Panc1. The basal level of PFKFB-4 protein expression is the highest in NUGC3 gastric cancer cell line and lowest in Panc1 cells, with the highest response to hypoxia in the pancreatic cancer cell line. Further studies showed that PFKFB-3 and PFKFB-4 gene expression was highly responsive to the hypoxia mimic dimethyloxalylglycine, a specific inhibitor of HIF-alpha hydroxylase enzymes, suggesting that the hypoxia responsiveness of PFKFB-3 and PFKFB-4 genes in these cell lines is regulated by the HIF transcription complex. The expression of VEGF and Glut1, which are known HIF-dependent genes, is also strongly induced under hypoxic conditions in gastric and pancreatic cancer cell lines. The levels of HIF-1alpha protein are increased in both gastric and pancreatic cancer cell lines under hypoxic conditions. However, the basal level of HIF-1alpha as well as HIF-2alpha mRNA expression and their hypoxia responsiveness are different in the MKN45 and NUGC3 cancer cells. Thus, the expression of HIF-1alpha mRNA is decreased in both gastric cancer cell lines treated by hypoxia or dimethyloxalylglycine, but HIF-2alpha mRNA expression is not changed significantly in NUGC3 and slightly increased in MKN45 cells. Expression of PFKFB-4 and PFKFB-3 was also studied in gastric cancers and corresponding nonmalignant tissue counterparts from the same patients on both the mRNA and protein levels. The expression of PFKFB-3 and PFKFB-4 mRNA as well as PFKFB-1 and PFKFB-2 mRNA was observed in normal human gastric tissue and was increased in malignant gastric tumors. The basal level of PFKFB-4 protein expression in gastric cancers was much higher as compared to the PFKFB-3 isoenzyme. In conclusion, this study provides evidence that PFKFB-4 and PFKFB-3 genes are also expressed in gastric and pancreatic cancer cells, they strongly respond to hypoxia via a HIF-1alpha dependent mechanism and, together with the expression of PFKFB-1 and PFKFB-2 genes, possibly have a significant role in the Warburg effect which is found in malignant cells.

Bobarykina AY ，Minchenko DO ，Opentanova IL ，Moenner M ，Caro J ，Esumi H ，Minchenko OH ... -  《ACTA BIOCHIMICA POLONICA》

Expression and hypoxia-responsiveness of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 in mammary gland malignant cell lines.

Minchenko OH ，Opentanova IL ，Ogura T ，Minchenko DO ，Komisarenko SV ，Caro J ，Esumi H ... -  《ACTA BIOCHIMICA POLONICA》

Hypoxic regulation of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene family (PFKFB-1-4) expression in vivo.

Minchenko O ，Opentanova I ，Caro J 《FEBS LETTERS》

Hyperinducibility of hypoxia-responsive genes without p53/p21-dependent checkpoint in aggressive prostate cancer.

Salnikow K ，Costa M ，Figg WD ，Blagosklonny MV ... -  《CANCER RESEARCH》