PKM1 and PKM2 represent two splice variants of the glycolytic enzyme pyruvate kinase thatdiffer in the inclusion of exon 9 or exon 10, respectively. PKM2 appears to be highlyexpressed in tumor cells and is suggested to favor the Warburg effect that is characterized byhigh glycolytic activity of tumor cells.In the first part of the thesis, we analyzed the regulation of PKM1 and PKM2 under normoxicand hypoxic conditions in different tumor cell lines from lung (A549) and prostate (PC3 andLNCaP). Furthermore, we investigated whether HIF-1alpha and HIF-2alpha are relevant for hypoxicregulation of PKM1 and PKM2.At the mRNA level, PKM1 and PKM2 were expressed in all cell lines studied. PKM2displayed significant upregulation under hypoxic conditions in all cell lines studied, whereasPKM1 mRNA was only increased significantly under hypoxic conditions in LNCaP cells. Atthe protein level, PKM2 expression was upregulated significantly in LNCaP cells in hypoxiaand to some extent in A549 cells that did not reach significance. No change of the PKM2protein level was observed in PC3 cells in hypoxia. Silencing of HIF-1alpha and HIF-2alpha did notshow any effect on PKM2 mRNA and protein expression levels in A549 and PC3 cells.However, HIF-1alpha dependent upregulation of PKM2 was observed at the mRNA and proteinlevel in LNCaP cells in hypoxia.Since HIF-1alpha is a mediator of the Warburg effect, we next analyzed a possible role of PKM2on the HIF-1alpha pathway. Interestingly, silencing of PKM2 decreased HRE activation in A549and PC3 cells, whereas HIF-1alpha protein levels were not significantly reduced after silencing ofPKM2, suggesting that HIF-1alpha dependence on PKM2 is mediated at the transactivation level.LNCaP cells responded differently in this regard, whereas no effect was observed aftersilencing of PKM2. Silencing of PKM1 increased HRE activation in these cells, an effect thatalso was observable only at the transactivation but not at the protein level of HIF-1alpha.PKM1 and PKM2 were further functionally characterized with respect to clonogenic survival.Silencing of PKM2, but not PKM1, significantly reduced clonogenic survival in all cell linesstudied, confirming the role of PKM2 in tumorigenesis in contrast to PKM1.In conclusion, we revealed that PKM2 was upregulated at the mRNA level in hypoxia in allcell lines studied. At the protein level, this effect became obvious only in LNCaP cells andwas dependent on HIF-1alpha in these cells. Vice verse, PKM2 appears to have an effect on HIF-1alpha transactivation in two from three cell lines studied. Thus, PKM2 may contribute to theWarburg effect by induction of HIF dependent signal transduction in at least some cancer celllines. PKM2 has strong effects on tumorigenesis to a similar extent in all cell lines studied.
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