Kras^G12D-LOH Promotes The Malignant Phenotype Of Pancreatic Ductal Adenocarcinoma Cells And Its Mechanism

[Background and Objective]Pancreatic cancer remains one of the most lethal types of solid malignancies,pancreatic ductal adenocarcinoma(PDAC)is the main pathologic type of pancreatic cancer.The occurrence and development of tumors is a complex process and multi-step and multi-genes were involved.Mutant Kras,particularly the point mutation in codon 12 of Kras(KrasG12D or KrasG12v)are frequently detected in PDAC.Furthermore,the wild-type allele status affect the response of cells to the activation of KrasG12D.Loss of heterozygosity(LOH)mainly occurs in tumor suppressor gene.However,mutant Kras with LOH was frequently detected in various tumours,the exact function and mechanism remain unclear.The occurrence and development of tumor is regulated by complicated signal network,especially the mammalian target of rapamycin(mTOR)plays an important role in regulating a wide range of functions,such as cell growth,proliferation and energy metabolism.Moreover,PDAC is insufficient in vascularity,in order to simulate hypoxia microenvironment in vivo,we investigated the impact of KrasG12D-LOH on the biologic behaviour and energy metabolism in PDAC cell under normoxic and hypoxic conditions,and further investigated that the effect of KrasG12D-LOH on mTOR signaling pathway.[Methods]1.Mouse pancreatic cancer cell lines 399(KrasG’2D PDAC cell lines)and 897(KrasG12D-LOH PDAC cell lines)were kindly gifted by the pancreas research centre of Technical University Munich.Cell lines were maintained in a humidified incubator(95%air and 5%CO2)at 37℃.Experiments under hypoxic condition were performed in the hypoxic incubator(1%O2,94%N2 and 5%CO2).The ability of cell proliferation and colony formation were determined by CCK-8 assay and Cell clone formation assay.2.Through the cell scratch test,we examined the impact of KrasG12D-LOH on PDAC cancer cell migration.Effect of KrasG12D-LOH on invasive activity of PD AC cells under normoxic and hypoxic conditions were detected by Transwell invasion assay.3.The cell cycle distribution and apoptotic rate were analyzed by flow cytometry.4.Intracellular adenosine triphosphate(ATP)content was measured using an ATP Assay Kit according to the manufacturer’s instructions.The supernatant was collected and lactate concentrations in the conditioned media were determined using a Lactate Assay kit according to the manufacturer’s instructions.5.The mRNA and protein expression level of Akt/Protein kinase B,AMP-activated protein kinase(AMPK),Regulated in development and DNA damage responses 1(REDD1),mammalian target of rapamycin(mTOR)were determined using qRT-PCR and Western blot analysis.[Results]1.The analysis results showed that the proliferation rate of 897 cells was significantly increased compared with 399 cells under normoxic or hypoxic conditions.We have observed significantly increased rate of colony formation by 897 cells when comparing with 399 cells under both normoxic and hypoxic conditions(P<0.05).These results indicated that KrasG12D-LOH can promote cell proliferation in PDAC cells.2.Using scratch test analysis,we found that the migration distances of cells of 897 cell was significantly higher than 399 cell under normoxia and hypoxia conditions.Transwell invasion analysis showed that the percentage of invasive cells was significantly higher in 897 cells compared with 399 cells(P<0.05 or P<0.01)under normoxic and hypoxic conditions.These results demonstrated that KrasG12D-LOH can promote metastasis in PDAC cells.3.Cell cycle progression and apoptosis of PDAC cells were investigated by flow cytometry.The results showed that the S phase ratio of 897 cells was higher than that of 399 cells(P<0.05)in normoxia.In hypoxia,cell cycle analysis also showed a dramatic increase in the number of cells in S phase(P<0.01),which was accompanied by a decrease in the percentage of cells in G1 phase of the cell cycle(P<0.05).However,the difference in the apoptotic between 399 and 897 cells was not statistically significant under normoxic or hypoxic conditions.4.Intracellular ATP content and lactate production were analysed to assess the glycolytic activity of 399 and 897 cells.The ATP concentration of 897 cells was significantly increased compared with 399 cells under both normoxic and hypoxic conditions(P<0.05 or P<0.01).The secretion of lactate in the supernatant of 897 cells was greater than that of 399 cells(P<0.05)under hypoxic conditions,whereas there was no significant difference in normoxia.These results suggested that KrasG12D-LOH enhances the glycolytic phenotype of PD AC cells(P>0.05).5.The results of qRT-PCR indicated that Akt,AMPK,REDD1 and mTOR genes were highly expressed in 897 cells compared with 399 cells under normoxic and hypoxic conditions.The result of Western blot shows that KrasG12D-LOH is linked with the up-regulated p-Akt,REDD1 and p-mTOR under normoxia and hypoxia.Furthermore,the phosphorylation level of AMPK was enhanced in KrasG12D-LOH cells under normoxia conditions.These findings suggested the possible involvement of mTOR signaling in the oncogenic function of KrasG12D-LOH.[Conclusions]1.KrasG12D-LOH is associated with increased cell proliferation,enhanced migration and invasion,progression from G1 to S phase under normoxic and hypoxiac conditions.2.KrasG12D-LOH is correlated with elevated intracellular ATP levels under normoxic and hypoxic conditions.In hypoxic,KrasG12D-LOH increased extracellular lactate production.3.The alteration of mTOR signaling pathway maybe a possible mechanism in the enhanced malignant phenotype and energy metabolism of KrasG12D-LOH PDAC under normoxic and hypoxic conditions.