Mohammad, GH; (2016) Pyruvate Kinase M2 (PKM2) and Lactate Dehydrogenase A (LDHA) as Novel Diagnostic Markers and Therapeutic Targets for Pancreatic Cancer. Doctoral thesis , UCL (University College London).

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Abstract

Pancreatic cancer is one of the most lethal malignancies worldwide; the early diagnosis of this disease remains challenging and there are few effective therapies, with palliative chemotherapy being the main treatment option for patients with locally advanced or metastatic disease. Alteration of cellular energy metabolism is one of the hallmarks of tumours. Therefore, I proposed that the study of metabolic enzymes such as pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA), which serve as key regulators of cellular energy metabolism in proliferating cells and mediators of aerobic glycolysis, could play an important role in the diagnosis and therapy of pancreatic cancer. Expression of PKM2 and LDHA was evaluated by immunohistochemistry in pancreatic cancer specimens. I found that the majority of PDAC strongly expressed PKM2 and LDHA at significantly higher levels compared with normal pancreatic tissues and benign pancreatic disease. PKM2 and LDHA expression directly correlated with tumour size and were expressed at higher levels in poorly differentiated tumours compared to well differentiated ones. Tumour cell proliferation, as detected by Ki67 staining, was significantly higher in tumours with strong PKM2 and LDHA expression compared to those with weak PKM2 and LDHA expression. Conversely, the number of CD8+ tumour infiltrating lymphocytes (TILs) was significantly higher in tumours with weak PKM2 and LDHA expression than in those with strong PKM2 and LDHA expression. Patients with tumours that had strong PKM2 and LDHA expression had a significantly worse overall survival compared with those that had weak PKM2 and/or LDHA expression (7.0 months vs. 27.9 months, respectively, p = 0.003, log rank test). Plasma PK and LDH concentrations were also significantly higher in pancreatic cancer patients compared to healthy controls (45.5 vs 21.6 U/L and 685 vs 194 U/L respectively, P < 0.0001). Shikonin (a Chinese herbal medicine) inhibited PKM2 activity and had a strong cytotoxic effect on pancreatic cancer cell proliferation with an IC50 of 2-3µM and 1-2 µM for 24 and 72 hours interaction time, respectively. Treatment of pancreatic cancer cell lines with combination of PKM2 activator IV (TEPP-46) and LDHA inhibitor (FX11) was synergistically inhibited pancreatic cancer cell proliferation, with combination indices (CI) of 0.48 and 0.45 for Miapaca-2 and BxPc-3 cell lines, respectively. Additionally, in the pancreatic cancer xenograft model, TEPP-46 and FX11 in combination significantly delayed both subcutaneous and orthotopic tumour growth compared to the control group (P ˂ 0.0001). The combination treatment also reduced expression of PKM2 and LDHA, and significantly decreased Ki-67 expression compared with controls (P ˂ 0.0001). Orthotopic xenografts treated with the combination therapy had a high number of CD8+TIL cells around the tumours. In conclusion, PKM2 and LDHA overexpression in pancreatic cancer is associated with poor outcome. As such, high expression of these two enzyme may contribute to the aggressiveness of pancreatic cancer and confer anergy against the host anti-tumour immune response. Tetramerisation of PKM2 in combination with inhibition of LDHA synergistically inhibited pancreatic cancer cell proliferation and growth both in vitro and in vivo and may represent a novel strategy for pancreatic cancer therapy.

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