Browsing by Subject "Receptor Protein-Tyrosine Kinases"
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Item Deciphering AXL-Driven Molecular Mechanisms of EMT(December 2021) Arner, Emily Nicole; Raj, Ganesh V.; Brekken, Rolf A.; Cobb, Melanie H.; Kim, JamesCellular plasticity, a feature associated with epithelial-to-mesenchymal transition (EMT), contributes to tumor cell survival, migration, invasion, and therapy resistance. Across human cancer, tumors that are high grade, poorly differentiated, and have undergone EMT carry a worse prognosis with a high likelihood of metastasis and poor outcome. AXL, a receptor tyrosine kinase (RTK), drives EMT and is implicated in tumor progression, metastasis, and therapy resistance in multiple cancer types including pancreatic cancer (PDA) and breast cancer. We investigated the contribution of TANK-binding kinase 1 (TBK1) to PDA progression and report that TBK1 supports the growth and metastasis of KRAS-mutant PDA by driving an epithelial plasticity program in tumor cells that enhances invasive and metastatic capacity. We identified that the receptor tyrosine kinase AXL induces TBK1 activity in a Ras-RalB-dependent manner. Furthermore, we report that AXL activation stimulates TBK1 binding and phosphorylation of the specific AKT isoform, AKT3 at S472. Activation of AKT3 drives the binding of AKT3 to slug/snail, where the complex is translocated into the nucleus. The binding of AKT3 to slug/snail protects the EMT-TFs from proteasomal degradation thus leading to an increase in EMT. These data suggest that the translocation of AKT3 to the nucleus is required for AXL-driven EMT and metastasis. Congruently, nuclear AKT3 expression correlates with worse outcome in aggressive breast. These results suggest that selective AKT3 targeting represents a novel therapeutic avenue for treating aggressive cancer that may avoid toxicity associated with pan-AKT inhibition. Additionally, our findings suggest that interruption of the AXL-TBK1-AKT3 cascade, has potential therapeutic efficacy in AXL positive metastatic cancer.Item Loss of TBK1 Kinase Function Improves Disease Outcome in Pancreatic Cancer and Metabolic Syndrome(2018-07-30) Cruz, Victoria Haley; Castrillon, Diego H.; Brekken, Rolf A.; MacDonald, Raymond J.; Scherer, PhilippAberrant expression and activity of TANK binding kinase 1 (TBK1) has been observed in numerous diseases. Here I've identified novel functions for TBK1 in pancreatic ductal adenocarcinoma (PDA) and in metabolic syndrome that promote disease progression. Activating mutations in KRAS are present in 90% of human PDA cases; yet direct pharmacological inhibition of K-RAS remains a challenge, indicating a need for effective therapies. Higher levels of TBK1 mRNA, a critical downstream mediator of oncogenic K-RAS in lung cancer, correlate with poorer outcome in PDA patients. Given these observations, I hypothesized that TBK1 is also an effector of K-RAS in PDA. KRAS mutant PDA cell lines are selectively sensitive to small molecule inhibition of TBK1. In K-RAS-driven genetic mouse models of PDA, Tbk1 supports spontaneous pancreatic tumor growth as evidenced by smaller tumors and fewer metastases in Tbk1 mutant PDA mice relative to normal PDA mice. Additionally, Tbk1 mutant tumors are more epithelial; an observation consistent with the reduced migratory phenotype of Tbk1 mutant tumor cell lines and lack of detectable metastases in Tbk1 mutant PDA animals. Mechanistic studies indicate that TBK1 is central to Axl-driven EMT and is activated with RAS in response to Axl stimulation in PDA cell lines. The latter part of this thesis is focused on the contribution of TBK1 to mice with metabolic disorder. TBK1 is implicated in the regulation of metabolism through studies with amlexanox, an inhibitor of IκB kinase (IKK)-related kinases. Amlexanox induced weight loss, reduced fatty liver and insulin resistance in high fat diet (HFD) fed mice and has now progressed into clinical testing for the treatment and prevention of obesity and type 2 diabetes. However, since amlexanox is a dual IKKε/TBK1 inhibitor, the specific contribution of TBK1 is unclear. To distinguish metabolic functions unique to TBK1, I examined the metabolic profile of global Tbk1 mutant mice challenged with HFD and investigated potential mechanisms for the improved metabolic phenotype. I report that systemic loss of TBK1 kinase function has a protective effect on metabolic readouts in HFD-fed mice, which is mediated by loss of an inhibitory interaction between TBK1 and the insulin receptor.Item Molecular Players in Lupus: Leads from Proteomic Screens(2014-05-23) Orme, Jacob Jennings; Yarovinsky, Felix; Davis, Laurie; Eagar, Todd N.; Monson, Nancy L.; Farrar, J. David; Mohan, Chandra; Satterthwaite, Anne B.Systemic Lupus Erythematosus is a multifactorial systemic autoimmune disorder marked by anti-nuclear antibodies (ANA), rashes and photosensitivity, joint inflammation, nephritis, and other clinical criteria. SLE develops through the breakdown of three major checkpoints: adaptive immune tolerance, peripheral innate responsiveness, and end-organ inflammation. Adaptive immune dysfunction produces autoantibodies leading to immune complex formation and deposition in the skin, joints, and kidneys. Innate immunity plays an important role in determining disease severity and progression. Molecular markers in patient blood and urine improve diagnosis and treatment of SLE. Proteomic screens identify such markers and provide important clues about disease pathogenesis. We have discovered that soluble Axl receptor tyrosine kinase, the Wnt/β-catenin pathway-related factors, and rare fibrinogen alpha chain variant A-α-E are elevated in the serum of patients with SLE. Here I explore these factors and their contributions to disease. I find that Axl tyrosine kinase is sheared from the surface of lupus-prone and SLE CD19+ and CD11b+/CD14+ leukocytes by proteases ADAM10 and TACE (ADAM17) to abrogate macrophage anti-inflammatory signaling through Twist. I further find that β-catenin is dysregulated in SLE but the deletion of β-catenin in lupus-prone macrophages does not appreciably change disease course. Lastly, I find that fibrinogen alpha chain isoform Aα-E may be associated with aPL-negative thrombotic complications in SLE.Item Pyrimidine Nucleoside Kinase UCK1 and TAM Receptor Tyrosine Kinase MerTK Converge on the Ubiquitin-Proteasome Pathway to Regulate EGFR(2014-04-14) Iwuaba, Veleka Cassie; Roth, Michael G.; White, Michael A.; Cobb, Melanie H.; Brown, Kathlynn C.Oncogenic addiction to EGFR is observed in many tumor types often as a result of gene amplification and/or activating mutations. In this study, we are following up on two hits from a kinase screen, Receptor Tyrosine Kinase MerTK and Pyrimidine Nucleoside Kinase UCK1. We have discovered, that in addition to perturbing EGFR signaling and accumulation, they converge on the ubiquitin-degradation pathway in NSCLC (Non-Small Cell Lung Carcinoma). Loss of UCK1 reduces EGFR accumulation by an EGF-independent mechanism but not other ErbB family members. Additionally, UCK1 depleted cells exhibit enhanced ubiquitin depletion, PARP inactivation, caspase-3 cleavage and increased BiP expression. Data from this study demonstrates that elevated BiP is likely due to depleted cytosolic ubiquitin pools and not induction of UPR. In contrast, Mertk loss results in significant EGFR accumulation, which appears to be enhanced by activating kinase mutations in EGFR, suggesting a trafficking defect in these cell lines. This data supports previous findings that EGFR mutants evade signal desensitization by prolonged residence in sorting endosomes and constitutive internalization/recycling.Item Regulation of Endocytic Recycling by FGD4, a Cdc42 GEF(2012-07-10) Cardenas, Jessica A.; White, Michael A.The family of Receptor Tyrosine Kinases (RTKs) are a group of cell surface receptors with the capability of activating, through phosphorylation, multiple kinase cascades in response to activation by an extracellular ligand. This allows a cell to respond to its environment and induce a range of cellular processes such as proliferation, differentiation, migration, and apoptosis. Unsurprisingly, these powerful transducers of extracellular signaling are often found mutated in human disease, such as cancer. Therefore, learning how these receptors are downregulated and processed once they have been activated may provide novel avenues of therapeutic intervention. How receptors are processed after internalization and fusion into the sorting endosome (also known as the early endosome) still largely remains unknown. Here, we discovered a Cdc42 GEF, FGD4, that may be important for shuttling ErbB receptors to the recycling endosome via a microtubule dependent mechanism. Through protein depletion studies we show that FGD4 is important for mitosis, microtubule stability, migration and endocytic trafficking of EGF, an ErbB1 ligand. Dynamic microtubule regulation are critical in these cell biological process, therefore we hypothesize that FGD4 may be regulating these diverse cell functions via a microtubule dependent mechanism.Item Validation of AXL as a Therapeutic Target in Pancreatic Cancer(2019-11-14) Du, Wenting; Malladi, Srinivas; Brekken, Rolf A.; Whitehurst, Angelique Wright; Malter, JamesPancreatic ductal adenocarcinoma (PDA), a leading cause of cancer-related death in the US, has a high metastatic rate that is associated with persistent immune suppression. AXL, a member of the TAM (TYRO3, AXL, MERTK) receptor tyrosine kinase family, has been identified as a critical factor that drives metastasis and immune suppression in many cancer types. Here we demonstrate that AXL-deficient PDA bearing mice have longer median survival, smaller tumors, fewer metastases and higher sensitivity to gemcitabine treatment compared to AXL wild-type (WT) PDA mice. AXL-deficient PDA displays a more differentiated histology, lower equilibrative nucleoside transporter 1 expression and has a more inflammatory and active immune microenvironment, all of which likely contribute to improved survival. As a result, AXL-deficient PDA treated with gemcitabine show more DNA damage (γH2AX) compared to WT PDA treated with gemcitabine. Single cell RNA sequencing of PDA genetically engineered mouse models reveals that AXL is expressed highly in tumor cells that have a mesenchymal-like phenotype and AXL expression correlates with classic markers of mesenchymal tumor cells. This AXL-positive mesenchymal PDA cell population is critical for PDA progression and metastasis, emphasizing the potential of AXL as a therapeutic target for pancreatic cancer patients. Multiple pharmacological strategies to inhibit AXL have been explored in pre-clinical models of PDA with promising therapeutic efficacy and similar phenotypes as genetically manipulation of AXL. These results provide a robust rationale for clinical studies aimed at investigating the effect of AXL inhibition in conjunction with standard therapy in pancreatic cancer patients.