Establishment Of Screening Models For Inhibitors Targeting IKKβ And MTOR Protein Kinases As New Anti-tumor Drugs

A potential link between inflammation and cancer has been suspected for over acentury, because recurring chronic inflammation and infection significantlyincreased the incidence of tumors, but the exact molecular mechanisms connectingthe two remain nebulous. In recent years, along with the continuously deepening theresearch, the IκB Kinase (IKK) complex and Mammalian Target of Rapamycin(mTOR) are gradually recognized as being vital in the course of chronicinflammation leading to cancer.In response to cell stimulation with TNFα, IKKβ phosphorylates IκBα (inhibitorof Nuclear Factor-κB) rapidly followed by degradation through the26S proteasome,which results in liberation of the NF-κB dimer which then translocates to the nucleusto activate gene transcription. Furthermore, IKKβ plays a critical role in couplinginflammation and cancer mainly through upregulating secretion of inflammatoryfactors. Over-expression or high-activity of IKKβ can be effective in stimulating thesecretion of inflammatory factors, such as TNFα, IL-1, IL-6, IL-8, CSF-1and COX2,etc. Inflammatory factors alter the microenvironment of tumor cell survival byregulating the interaction between tumor cells and stromal cells, as well as tumorcells and extracellular matrix, endogenously promote tumor growth, invasion andproliferation.Several mTOR-regulated processes contribute to uncontrolled growth in bothmalignant and benign tumors, such as stimulating protein and lipid synthesis,glycolytic gene expression, the pentose phosphate pathway, mitochondrialbiogenesis and oxidative metabolism. Multiple signaling pathways may affect tumorgrowth and progression by modulating mTOR activity. Growth factors, mitogens,energy, nutrients, and cellular stress influence mTOR activity through posttranslational modification (e.g., phosphorylation) of tumor suppressors TSC1and TSC2. Numerous kinases, including AKT, RSK1, ERK, GSK, CDK1, p38, MK2and AMPK have been suggested to function as TSC kinases that integrateextracellular signals and tumorigenesis through control of the TSC1/TSC2/mTORpathway. Wnat’s more, inflammatory cells and factors contribute to tumordevelopment, for tumor-associated macrophages (TAMs) release TNFα and IL-1β,significantly associated with reduced relapse-free and overall survival rates in manyhuman cancers, supporting that the main function of TAMs is tumor promotion, byinducing cancer cells to produce mTOR-mediated other factors, such as VEGF.More to the point, activated IKKβ also phosphorylates TSC1, alters itsmembrane localization and inhibits its association with TSC2, thereby activating themTOR pathway, enhancing VEGF expression, angiogenesis and tumorigenesis. In aword, inflammatory cytokine TNFα is able to activate the mTOR pathway andenhance angiogenesis via IKKβ-mediated suppression of TSC1.Therefore, in addition to illustrating the critical roles of IKKβ and mTORmolecules in linking inflammation and cancer and providing an explanation forincreased cancer risk in response to persistent infections and inflammation, thesetheories also identify new targets for development of novel anti-cancer therapies andpreventive strategies. For instead of targeting the cancer cell itself, as done byconventional anti-cancer drugs, the new therapeutics will target processes that occurwithin inflammatory cells that are essential for cancer development and progression.Unlike cancer cells, inflammatory cells retain a normal and stable genome andtherefore are unlikely to become genetically resistant to therapeutic intervention.This paper aims to establish kinase inhibitors screening model which can beused to screen for specific IKKβ and mTOR inhibitors. Through molecular biologymethods we tried prokaryotic expression systems and baculovirus expression systemto prepare exogenous protein IKKβ and mTOR. Comparing activities of bothexpression products, we decided to utilize the Bac-to-Bac Baculovirus ExpressionSystem, and built donor plasmid and shuttle plasmid of IKKβ and mTOR FRB andKinase Domain truncation mutant mTOR-FKD. At last, expressed by viral infection of insect cells and purified by affinity chromatography, IKKβ and mTOR-FKD wereobtained successfully. Based on the substrate phosphorylation reaction by kinasehydrolyzing ATP in vitro, we used fixed factors method, optimizing theconcentration of ATP, substrate and kinase in the kinase reaction system one by one.Also it is essential to evaluate the screening model by positive inhibitors, in order toensure the stability and sensitivity. Supported by the bioluminescence principle,Kinase-Glo Luminescent Kinase Assay was adopted as the testing method toestablish the molecular level serine/threonine protein kinase IKKβ and mTORinhibitor screening model, providing an effective tool to find the specific inhibitorstargeting the kinases in the future.