Investigating The Role Of PKM2 And The Potential Of PKM2 As A Therapeutic Target Of Oncolytic Adenovirus In Pancreatic Cancer Treatment

The M2 isoform of pyruvate kinase?PKM2?is a key driver of glycolysis in cancer cells and has critical“non-metabolic”functions in some cancers.The potential of PKM2 as a therapeutic target was widely studied in various cancers.The treatment of tumors with targeting PKM2 is focused in various cancers.As a cancer with metabolic disorder,pancreatic cancer patients has poor prognosis for the lack of durable treatment.It is critical to identify novel therapeutic targets and develop potential therapeutic strategies for pancreatic cancer.However,the role of PKM2 in pancreatic cancer remains unclear.In the current study,we used Immunohistochemistry?IHC?,Western blotting,qRT-PCR,CCK-8,cell counting et al.to elucidate the role of PKM2 in pancreatic cancer progression.What's more,an oncolytic adenovirus carrying shPKM2(O^Ad.R.shPKM2)was constructed to test the potential of PKM2as a therapeutic target in pancreatic cancer cell lines and PANC-1 xenografts.The mechanism of O^Ad.R.shPKM2 was studied on apoptosis and autophagy.The results are following:1.PKM2 expression in the pancreatic cancer tissues was significantly up-regulated compared with that in the adjacent noncancerous tissues by IHC assay.We further assessed the correlation between PKM2 expression and clinical characteristics of pancreatic cancer patients and found that PKM2 expression was correlated with worse overall survival in these pancreatic cancer patients.Additionally,PKM2 expression was higher in several pancreatic cancer cell lines than in the normal pancreatic duct cell line.2.We constructed PKM2 knockdown cells by transducing pancreatic cancer cells with lentivirus containing PKM2-targeted shRNA.PKM2 knockdown significantly inhibited cell viability as measured by the CCK-8 and cell counting assays.Cell migration in pancreatic cancer cells with PKM2 knockdown was suppressed.What's more,silencing PKM2significantly inhibited tumor growth.3.Knockdown of PKM2 in pancreatic cancer cells caused a significant decrease in the LC3II/LC3I ratio and Beclin-1 expression in conjunction with P62 accumulation.The number of LC3 puncta was reduced in cells with silenced PKM2.A small number of autophagic structures were found in PKM2-knockdown cells,and the mRNA and protein expression levels of the autophagy-related transcription factors HIF-1?and FoxO3a were both reduced in PKM2-knockdown cells,suggesting PKM2 knockdown inhibited cell growth by suppressing autophagy.4.We constructed an RGD-modified oncolytic adenovirus carrying PKM2-targted shRNA(O^Ad.R.shPKM2)to investigate whether PKM2 could be a therapeutic target for pancreatic cancer.O^Ad.R.shPKM2 specially down-regulated PKM2 expression in pancreatic cancer cells and showed potent cytotoxic effect on pancreatic cancer in vitro and in vivo.Additionally,O^Ad.R.shPKM2 promotes cell apoptosis and inhibits cell autophagy in pancreatic cancer.In this study,we observed that PKM2 is highly expressed in patient's samples with pancreatic cancer and is correlated to survival.Elevated PKM2 expression promoted cell proliferation,migration and tumor formation.The inhibition of cell growth by silencing PKM2 is caused by impairment of the autophagy process.To test the potential effects of down-regulating PKM2 as a clinical therapy,weconstructed an RGD-modified oncolytic adenovirus containing shPKM2 to knock down PKM2 in pancreatic cancer cells.Cells transduced with O^Ad.R.shPKM2 exhibited decreased cell viability,and in a PANC-1 xenograft model,intratumoral injection of O^Ad.R.shPKM2resulted in reduced tumor growth.Furthermore,O^Ad.R.shPKM2 induced apoptosis and impaired autophagy in PANC-1 cells.Our results suggested that targeting PKM2 with an oncolytic adenovirus produced a strong anti-tumor effect and that this strategy could broaden the therapeutic options for treating pancreatic cancer.