Question

This discussion focuses on the regulation of glycolysis and gluconeogenesis by phosphofructokinase-2 and will help you apply your understanding of these pathways and their regulation to adaptations in cancerous cells.

You have successfully completed your internship rotation with the antibiotic group at MethylTranspharmiX and have moved into their Cancer Therapeutics division.

In many cancers, cells use aerobic glycolysis rather than oxidative phosphorylation as their main energy source. This is known as the Warburg effect, and was first described by Otto Warburg in 1924, who observed that cancer cells have a much higher rate of glycolysis and a lower rate of respiration compared to normal cells. He observed that cancer cells have adapted to low oxygen conditions inside solid tumors by not needing oxidative phosphorylation to generate ATP, and that the cancer cells shut down the mitochondria.

In your position at MethyltranspharmiX, you are studying a gene upregulated in tumor cells, an isoform of PFK2, a bifunctional enzyme that has both kinase and phosphatase activities. Isoforms are genes that express proteins that have a similar structure and function, but have slight variations, giving the proteins unique properties that are often tissue specific. In humans there are four different genes for PFK-2, encoding four different isoforms. The genes are all PFKFB (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) isoforms. The four isoenzymes of PFK2 include the liver-specific version: PFKFB1, one found in the heart: PFKFB2, one that is expressed in the placenta and is inducible in low oxygen conditions, PFKFB3, and finally PFKFB4 a testes specific isoform, which is also essential to cancer cell survival. In cancerous cells, genes are frequently turned “on” or “off”, changing when and where the gene products are being expressed. This is how the cancer cells form tumors, metastasize and grow in new locations, as well as grow quickly.

Kinase activity

Bisphosphatase activity

Fructose 6-phosphate ATP Kinase Bisphosphatase activity activity ADP Fructose 2,6-bisphosphate

Figure 1 – Summary of the two catalytic activities of the PFKFB isozymes. Kinase activity of PFKFB genes produces fructose 2,6-Bisphosphate by addition of a phosphate to fructose 6-phosphate. Bisphosphatase activity removes a phosphate from fructose 2,6-bisphosphate to produce fructose 6-phosphate. Figure adapted from Lehninger Principals of Biochemistry, fifth edition.

The adenocarcinoma tumor cells you are studying have been found through a cDNA microarray to have high expression levels of one of the PFK2 isoforms, the gene product of PFKFB3 known as iPFK2.   Your supervisor suspects that iPFK2 enzyme has unique properties that may allow cancer cells to change their regulation of glycolysis and gluconeogenesis. Your team is investigating this enzyme as a potential cancer chemotherapy target.

In the lab, you have been working to determine what makes iPFK2 unique by characterizing both the kinase and phosphatase enzyme activity for the enzyme as well as isoforms purified from the liver, heart, skeletal muscle, testes and brain.

Table 1: Comparison of the kinase activity properties of the various Fru-6-P,2-Kinase/Fru-2,6-BPase isozymes:

Kinase:	iPFK2 Placental	Liver	Heart	Skeletal Muscle	Testes	Brain
Vmax (mU/mg)	142	57	61	66	90	90
Fru-6-P Km (uM)	32	16	74	56	85	27

  

Table 2: Comparison of the bisphosphatase activity properties of the various Fru-6-P,2-Kinase/Fru-2,6-BPase isozymes:

Bisphosphatase:	iPFK2 Placental	Liver	Heart	Skeletal Muscle	Testes	Brain
Vmax (mU/mg)	0.2	45	10	154	22	29
Fru 2,6-BisP Km (uM)	130	0.5	40	0.4	21	70

Table 3: Comparison of the activity ratio of Kinase to bisphosphatase activity for the various Fru-6-P,2-Kinase/Fru-2,6-BPase isozymes:

Activity Ratio	iPFK2 Placental	Liver	Heart	Skeletal Muscle	Testes	Brain
Kinase/bisphosphatase	710	1.3	6.1	0.4	4.1	3.1

For your initial post discuss the following questions: 1) Based on the kinase and bisphosphatase activities listed above for the different enzymes, what differences are observed in the iPFK2 enzyme compared to other isozymes? Consider both the kinase and bisphosphatase activities, as well as the relationship between the two. 2) What would we expect to happen in the adenocarcinoma cells which have high expression of iPFK2? 3) How would it change carbohydrate metabolism? Be sure to explain your answer. 4) How is this different from the other PFK2 enzymes?

Answer 1

1. By the analysis of the above data, we concluded that the iPFK2 enzyme i.e, the gene product of PFKFBF3 gene present in the placenta have more kinase activity and low bisphosphatase activity. The placenta cell are differentiated and proliferated under low oxygen condition, like cancerous cells, however, they require more energy (ATP) molecules. Therefore, the glycolysis is pathway is active and respiration rate is low. As result of it kinase activity of iPFK2 is much more than other organ/tissue cells.

2. Adenocarcinoma cells are high dividing and proliferated in uncontrolled manner. They have high iPFK2 expression because they will grow under less oxygen (low respiration rate) supply but they require more energy (ATP) utilized by the aerobic glycolysis pathway. Therefore for the more kinase activity of the enzyme should be necessary to proceed the metabolic pathway.

3. Carbohydrate molecules are metabolized rapidly in case of iPFK2 cells- placenta and cancer. This must be required for the pay-off (ATP formation) pathway of glycolysis. However in case of other cells that have more mitochondria bisphosphatase activity is more than the kinase activtiy of the PFK2 isoforms.

4. The PFK 2 enzyme isoforms for the different tissue/ organs are vary based upon the in situ condition of the cells. The cell have more aerobic condition or oxygen supply, have more oxidative phosphorylation i.e., bisphopshatase activity for the ATP generation and large number of mitochondria. However the cells are under stressed condition have more kinase activity for the utilization of energy source.

In case of cancer and placenta cells the kinase activity of PFK2 isoform is more than the bisphosphtase. But in case of skeleton muscle cell faster ATP generation is required therefore, more oxygen is need, more carbohydrate metabolism, large number of mitochondria and more bisphosphatase activity.