BRSK2 Gene Enhances The Drug Sensitivity Of Pancreatic Ductal Carcinoma Cells And Its Involvement In Endoplasmic Reticulum Stress Induced Apoptosis

Endoplasmic reticulum is a very important organelle in eukaryotic cells where cells deal with folded proteins. It is also a place where newly synthesized peptides are modificated to form the correct tertiary structure. When proteins in endoplasmic reticulum cannot be handled properly, endoplasmic reticulum stress happened. By activated pathways downstream in order to activate unfolder protein response (UPR), ER stress protects cells from damage. ER stress and UPR have been implicated in diabates, tumors, neurodegenerative diseases and cerebral ischemia.VCP, which is also called p97 in mammals, is a member of ATPases associated with various cellular activities (AAA) family. VCP is also called melanin transferrin, has a molecular weight of 97.2KD. Many protein pathways in cells contain VCP. In ER stress, vcp acts as a member of endoplasmic reticulum-associated degradation (ERAD) to take part in ERsBRSK2 was a new gene which was first cloned by our lab. Researches about BRSK2 were focus on polarization of nerve, formation of axons and control of cell cycles. Preliminary works which were done in our lab before pointed out that BRSK2 play an important role in tolerance of PANC-1 to glucose starvation. But until now, no reports about its parts in ER stress. Here we first report that BRSK2 is activated in ER stress in both transcription and translation ways by using ERs drugs. These act as transcription and translation level improved. What’more, the subcellular localization of vivo BRSK2 were changed during ERs drugs, and BRSK2 can cofocus with ER protein Herp.When vivo BRSK2 were knockdown down in HeLa cells, apoptosis induced by ERs drugs are signifigated more than control groups.It had been reported that another member of AMPK family called SIK2, can activate VCP to regulate ERAD. If BRSK2 also can control activication of VCP to regulate ERAD?Before experiment, we test several cancer cells using western-blot and found that many cancer cells have vivo VCP express.In which hepatoma cells and pancreatic cancer cell PANC-1 have a higher expression of endogenous VCP, what’s more PANC-1 cell also has expression of endogenous BRSK2. So we choose PANC-1 cell to do vivo experiments.Confocus experiment also proved that BRSK2 and VCP have vivo con-location in cells in glucose deprevation.First of all, vivo and in vitro interaction experiments demonstrated that BRSK2 and VCP interacted with each other. Then in order to clarify BRSK2 connects which domain of VCP, we construct prokaryotic expression vector which contain different fragments protein of VCP and by using GST-pull down experiment, we proved that BRSK2 can connect with VCP three protein fragments except D1 domain.Among these three connections, combination with the N-terminal is most closely.Later, in HeLa cell transfacted with exogenous VCP, we found that BRSK2 can co-location with VCP in both normal and ERs drugs treated cells.In conclusion, we found that BRSK2 is activated in ERs, knockdown of BRSK2 can cause an improvement of apoptosis cells by ERs drugs in HeLa cells compared with control group which BRSK2 were not knock down. This phenomenon may be due to interaction between BRSK2 and VCP.Pancreatic ductal adenocarcinoma (PDAC) which is also known as pancreatic cancer is a malignant cancer of the digestive system. PDAC is formed from deterioration of pancreatic epithelial cells. The death cause rate of PDAC is No.4 in China. Pathogenesis of pancreatic cancer is not very clear until now. The only direct cause of PDAC known is smoking. Because there is no obvious symptoms in the early stages of PDAC, when jaundice phenotype can be observed is already in the late stages of PDAC, so PDAC is hard to treat. Highly malignant, metastasis and strong invasion make is more difficult to treat with. So the 5-year survival rate is as low as less than 5% for PDAC.BRSK2 is a new gene which first cloned in our lab. Early researchers thought it only expressed in brain, so was given name called brain-specific kinase 2,BRSK2.But later, people found that is not only expressed in brain but also in pancreas. Early work in our laboratory found that BRSK2 is in a special high expression in pancreatic cancer cell PANC-1; further studies have shown that BRSK2 is an important factor of tolerance of PANC-1 for glucose starvation. Expression and kinase activity of BRSK2 are both uploaded in glucose starvation. Molecular mechanisms of BRSK2’s regulation for PANC-1 cells in glucose starvation are through phosphorylation of TSC2, high expression of BRSK2 can negatively regulate mTOR pathway and inhibit protein synthesis. What’s more, over expression of BRSK2 may cause cell cycle arrest in G2/M phase; this may slow down cell division before surrounding environment get better. We’ve known that pancreatic cancer cell has a strong tolerance to harsh environment, which makes it hard to treat. While our data shown that BRSK2 is an important factor of tolerance of pancreatic cancer cell for glucose starvation, I follow this to research that if BRSK2 can be a drug target for treatment of pancreatic cancer cell.First, by use RNAi technology, we constructed stable cells lines of PANC-1 with stable knockdown expression of BRSK2 and use it for screen chemotherapy drug. We found that stable cells with down expression of BRSK2 are more sensitive to daunorubicin, doxorubicin, mitomycin and rapamycin. What’s more, they have a small IC50 than control group. Similar results were obtained by use two different target siRNA in transient transfection. In another pancreatic cancer cell Miapaca-2, which also has Endogenous BRSK2, we got the same results.By using FACS test apoptosis cells in Sub-G1 method, we found that in low-dose daunorubicin (0.5μM) treatment, PANC-1 cells which BRSK2 were knockdown had more apoptosis cells than control group which BRSK2 were not knockdown. Combined with previously obtained molecular mechanisms of BRSK2, we concluded that in PANC-1 and Miapaca-2 cells which p53 protein function are missing, functions of DNA damage only cycle checkpoint were disturbed and cells could not damage to self-repair for un arrest of cell G2/M cycle. Finally, knockdown BRSK2 cause cells more sensitive to chemotherapy.Autophagy is a common physiological and pathological process of living, autophagy caused by drugs often leads to apoptosis even death in pancreatic cancer cells. We found that rapamycin can cause enhancement of autophagy in pancreatic cancer cells PANC-1 and knockdown of BRSK2 can make autophagy degree of PANC-1 cells caused by rapamycin increase. But knockdown of BRSK2 did not cause level of ROS change in PANC-1 cells, this show that BRSK2 does not through change effect of ROS to participate in autophagy.Combined usage with gemcitabine and AMPKI, we found that growth of PANC-1 cells were significated slow than control group. But when use AMPKI in nude mice, we found that animals were dead. There are black phenomenon in liver, stomach, intestines and other organs in the Anatomical dead nude mice. We thought that because insulate in water and high cytotoxic made AMPKI unfit for use as a drug.In conclusion, we found that knockdown of BRSK2 in p53 defective pancreatic cancer cells causes increased sensitivity to daunorubicin, doxorubicin, mitomycin and rapamycin. This because that expression of BRSK2 can control cell cycle by arrest it at G2/M. By knockdown of BRSK2, p53 defective cancer cells cannot protect itself under DNA damage caused by chemotherapy.