Clinical review and experimental evaluation of tumour M2-pyruvate kinase in pancreatic cancer

Abstract

The treatment of pancreatic cancer is challenging. Patients are often beyond curative surgical therapy and palliative treatment with chemotherapy provides limited benefit. New markers of cancer activity and therapeutic targets are required. This thesis has firstly reviewed the available literature on Tumour M2-PK, a dimeric form of M2 isoenzyme of pyruvate kinase, in GI cancer and carried out a meta-analysis of the clinical data on pancreatic cancer. Experimental work evaluated the measurement of M2-pyruvate kinase in human pancreatic cancer cell lines with altered microenvironment (hypoxia, acidic pH or glucose-deprived condition). Tumour M2-PK level was measured using ELISA, total M2-PK by immunoblotting and pyruvate kinase activity by spectrophotometric analysis. Apoptosis or necrosis was detected by measuring active Caspase 3/7 and 8, Bcl-2, Bax and Annexin V staining. Localisation of M2-PK in pancreatic cancer cell was studied by immunocytochemistry. The clinical review has shown that Tumour M2-PK is not an organ-specific marker of GI cancer but is elevated with positive predictive value of 86–88% in gastro-oesophageal and colorectal cancers. In pancreatic cancer the diagnostic odds ratio (DOR) of an elevated Tumour M2-PK was similar to those of CA19-9 with overall sensitivity of 94% and specificity of 55%. Higher levels of Tumour derived M2-pyruvate kinase were observed in Colo 357 cell lines compared to Panc-1 cells. Exposure of Colo 357 cells to altered culture conditions resulted in decreased cell proliferation accompanied by elevated Tumour M2-PK levels with unchanged total M2-PK levels suggesting tetramerdimer switch-over, which was confirmed by the corresponding change in the pyruvate kinase activity. No correlation of Tumour M2-PK level or PK activity with apoptotic or anti-apoptotic markers was observed. Immunocytochemistry suggested M2-PK localisation to intracellular membrane-bound structures with no translocation to nucleus or mitochondria under altered tumour microenvironment. Conclusion: Tumour M2-PK is a potential marker of pancreatic cancer. Altering the tumour microenvironment causes a switch to measured M2-PK levels. This may allow cells to overcome cell apoptosis and could be a pathway facilitating tumour survival