Brsk2 Tolerance Adjustment Mechanism In Pancreatic Ductal Carcinoma Cell Energy Pressure

Tumorigenesis is one of the most important fields in oncobiology. As tumors growand invade beyond their homeostatic limits, the tumors are subjected to insufficientglucose, oxygen, nutrition factors and insufficient vascularization. Some malignanttumors exhibit extensive growth, invasion, and metastasis in these environments.Pancreatic duct cancer line, PANC-1, is the represent of these malignancies. Ourresearch indicated that the tolerance of PANC-1 to glucose starvation wassignificantly higher than that of other cancer cell lines. We proposed that thistolerance might be a key factor in tumor progress.The gene, expressed in brain and pancreas specifically, is cloned firstly by us andencodes the protein which is classified to AMPK subfamily of CAMK protein kinasefamily. Since the gene was detected in brain specifically in the past, it was namedBRSK2 (Brain Selective Kinase 2). The results of Northern blot and western blotindicated that BRSK2 was highly expressed in brain and pancreas specifically, and theexpression in pancreas is higher than that in brain. We detected the expression ofBRSK2 in many cancer cell lines and found it was expressed in PANC-1 specifically.The experiment showed that BRSK2 had no effect on cell proliferation of PANC-1 butcould significantly enhance the cell survival of PANC-1 under glucose starvation,while knockdown of BRSK2 by RNAi strongly decreased the tolerance of PANC-1 toglucose starvation. There results showed that BRSK2 might be the key factor of thistolerance. Further investigation showed that BRSK2 interacted with TSC2 in vitro andBRSK2 phosphorylated TSC2 on S1387. It is known that TSC2 functions as a keyplayer in regulation of the mTOR pathway, and the phosphorylation state of TSC2affects the activity of mTOR pathway. So we further investigated the relationshipbetween BRSK2 and mTOR, and we found that as the level of BRSK2 increasing, theactivity of mTOR and its substrates S6K and 4EBP1 decreased. This result wasproved by three experiments including overexpression BRSK2, energy stress inducinggradient experiment and siRNA interference experiment. Besides that, we found knockdown of BRSK2 by RNAi blocked glucose starvation induceddephosphorylation of S6K, and we also found BRSK2 reduced the activity of S6Kthrough TSC2. The flow cytometry analysis showed that BRSK2 enhanced thetolerance of PANC-1 to glucose starvation through TSC2. Further investigationshowed that overexpression S6K increased the rate of cell death, and coexpression ofBRSK2 with S6K could rescue the S6K-mediated cell death. We expressed the mutantBRSK2KD in PANC-1 cell with gradient, because of the BRSK2KD competition withendogenous BRSK2, the phosphorylation activity of S6K was increased along withthe expression level of BRSK2KD, and also following the decrease of survival cellswith gradient.In this study, we found BRSK2 enhanced the tolerance of PANC-1 to glucosestarvation through TSC2/mTOPUS6K pathway. As we all know, SrK and 4EBP1 playan important role in the regulation of translation initiation. Because of the inhibitionof activity of S6K and 4EBP1 by BRSK2, the translation initiation of some proteinwas block, and energy expenditure was decreased, thus, the cells could be survivalunder glucose starvation. In a word, we believed that BRSK2 may play an importantrole in tolerance of PANC-1 to glucose starvation. These results may help usinvestigate the pancreatic cancer progression and find the target for therapeutic drugdesign to pancreatic cancer.In this study, we also identified and characterized two new human genes. Whenwe investigated the mTOR pathway, we cloned a novel member of Ras family namedRHEBL1, belonging to the Rheb branch of small GTPase proteins. The cDNAsequence contains an open reading frame of 551 bp, encoding a putative protein of183 amino acid residues. The expression pattern of RHEBL1 showed that it wasubiquitously expressed in 17 tissues. RHEBL1 gene encodes a 20.69 kDa protein,localized in cytoplasm when overexpressed in COS7 cells. Reporter gene assaysshowed that overexpression of PHEBL1 in HEK 293T cells strongly activated thetranscriptional activities of NF-kappa B, while the mutant (D60K) only weaklyactivates NF-kappa B-mediated transcription. Our findings suggest that RHEBL1 is apositive regulator of NF-kappa B-mediated gene transcription. And in the futureresearch, we will investigate whether RHEBL1 is related with mTOR pathway.Moreover, we report here the cloning and characterization of the human geneDERP6, isolated from human testis cDNA library, and mapped to 17p13.1 by searching the UCSC genomic database. The DERP6 cDNA consists of 1486nucleotides and has a 316-amino acids open reading frame. The Northernhybridization analysis showed that DERP6 was ubiquitously expressed in all the 16adult tissues, especially highly expressed in heart, brain, liver, skeletal muscle andtestis. DERP6 protein was located in cytoplasm when overexpressed in cultured cells.Reporter gene assays showed that overexpression of DERP6 in cells activated thetranscriptional activities of p53. These results indicate that the human gene DERP6may be involved in p53-mediated gene transcription. While, how DERP6 affected thep53 pathway? Did DERP6 affect the cell cycle and apoptosis through p53 pathway?We will answer these questions in the future research.In conclusion, we found BRSK2 enhanced the tolerance of PANC-1 to glucosestarvation through TSC2/mTOR/S6K pathway. Then we believed that BRSK2 mayplay an important role in tolerance of PANC-1 to glucose starvation. These resultsmay help us investigate the pancreatic cancer progression and find the target fortherapeutic drug design to pancreatic cancer. And the identification andcharacterization of mTOR-related and p53-related gene RHEBL1 and DERP6 alsoprovide new material in cell survival research.