Browsing by Subject "Carcinoma, Pancreatic Ductal"
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Item Fibulin-5 Promotes Pancreatic Tumor Growth through Inhibition of Integrin-induced ROS: Insights into Tumor-Matrix Signaling(2016-07-18) Topalovski, Mary; Terada, Lance; Cobb, Melanie H.; Abrams, John M.; Brekken, Rolf A.Elevated oxidative stress is an aberration seen in many solid tumors, and exploiting this biochemical difference has the potential to enhance the efficacy of anti-cancer agents. Homeostasis of reactive oxygen species (ROS) is important for normal cell function, but excessive production of ROS can result in cellular toxicity and therefore ROS levels must be balanced finely. Here, we highlight the relationship between the extracellular matrix and ROS production by reporting a novel function of the matricellular protein Fibulin-5 (Fbln5). We found that Fbln5 is abundantly expressed in mouse and human pancreatic cancer compared to normal pancreas. By employing genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDA), we showed that mutation of the integrin-binding domain of Fbln5 led to decreased tumor growth, increased survival, and enhanced chemoresponse to standard PDA therapies. Through mechanistic investigations, we found that improved survival was due to increased levels of oxidative stress in Fbln5 mutant tumors. Furthermore, loss of the Fbln5-integrin interaction augmented fibronectin (FN) signaling, driving integrin-induced ROS production in a 5-lipooxygenase-dependent manner. These data indicate that Fbln5 promotes PDA progression by functioning as a molecular rheostat that modulates cell-ECM interactions to reduce ROS production and thus tip the balance in favor of tumor cell survival and treatment-refractory disease. The latter part of this thesis is focused on the underlying mechanism that leads to upregulation of Fbln5 in PDA. The deposition of ECM is a defining feature of PDA where ECM signaling can promote cancer cell survival and epithelial plasticity programs. ECM-mediated signaling is governed by expression of the ECM proteins, the presence of cell surface receptors and the expression and activity of matricellular proteins that function as extracellular adaptors to reduce ECM-cell interaction. As stated above, Fbln5 is a matricellular protein that blocks FN-integrin interaction and thus directly limits ECM-driven ROS production and supports PDA progression. Compared to normal pancreatic tissue, Fbln5 is expressed abundantly in the stroma of PDA; however, the mechanisms underlying the stimulation of Fbln5 expression in PDA are undefined. Using in vitro and in vivo approaches, we report that hypoxia triggers Fbln5 expression in a transforming growth factor β (TGF-β)- and PI3K-dependent manner. Pharmacologic inhibition of TGF-β receptor (TGF-βR), PI3K, or protein kinase B (AKT) was found to block hypoxia-induced Fbln5 expression in mouse embryonic fibroblasts and 3T3 fibroblasts. Moreover, tumor-associated fibroblasts from mouse PDA were also responsive to TGF-βR and PI3K/Akt inhibition with regard to suppression of Fbln5. In genetically engineered mouse models of PDA, therapy-induced hypoxia elevated Fbln5 expression while pharmacologic inhibition of TGF-β signaling reduced Fbln5 expression. These findings offer insight into the signaling axis that induces Fbln5 expression in PDA and a potential strategy to block its production.Item Inhibition of Discoidin Domain Receptor 1 Reduces Collagen-Mediated Tumorigenicity in Pancreatic Ductal Adenocarcinoma(2015-07-24) Aguilera, Kristina Yolanda; Pearson, Gray W.; Brekken, Rolf A.; Minna, John D.; Terada, LanceAn extracellular matrix (ECM) rich in fibrillar collagens is a principal component of pancreatic ductal adenocarcinoma (PDA). The ECM provides structural support for the tumor and facilitates tumor cell survival and chemoresistance by activating cell surface receptors on tumor cells. Fibrillar collagens bind the collagen-specific receptor tyrosine kinase discoidin domain receptor 1 (DDR1), implicated in regulating cell proliferation, migration, adhesion, ECM remodeling, and response to growth factors. Additionally, collagen has been shown to promote chemoresistance in pancreatic tumor cells. I hypothesize that the regulation of collagen-mediated DDR1 signaling promotes chemoresistance. Collagen expression and deposition is a complex process that is orchestrated in part by the matricellular protein SPARC. SPARC expression in human PDA patients correlates with improved chemoresponse; however, the mechanism underlying this is unclear. I proposed that SPARC reduces collagen binding to collagen receptors. Structural studies identified that SPARC and DDR1 share the same collagen-binding site. I demonstrated that SPARC inhibited collagen binding to DDR1 via in vitro binding assays and cell-based activity assays. To determine the functional relevance of Sparc expression and collagen-mediated Ddr1 activation in PDA, Sparc-null (Sparc-/-) mice were crossed with a GEMM of PDA, KIC (LSL KrasG12D/+; Ink4aArflox/lox; p48Cre/+). Survival was reduced and tumors were more aggressive in Sparc-/-; KIC mice. Tumors from these animals also displayed elevated Ddr1-mediated signaling. Human PDA, and primary PDA cell lines isolated from Sparc+/+; KIC and Sparc-/-; KIC animals, were used to probe collagen signaling and collagen activation of DDR1 stimulated downstream intermediates including protein tyrosine kinase 2 (PYK2) and pseudopodium-enriched atypical kinase 1 (PEAK1). Furthermore, utilization of a novel DDR1 small molecule inhibitor (7rh) abrogated collagen-induced DDR1 signaling and blunted tumor cell colony formation, migration, and enhanced sensitivity to gemcitabine. Additionally, 7rh inhibited Ddr1 signaling in syngeneic, genetic, and human xenograft pancreatic tumors and was well tolerated. Therapy studies combining standard chemotherapy (gemcitabine plus nab-paclitaxel) with 7rh in vivo dramatically improved survival of mice compared to standard therapy alone. These data confirm that inhibition of collagen signaling in PDA is an attractive therapeutic strategy and demonstrate that DDR1 is a target that can be inhibited pharmacologically.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 Targeting Cyclin Dependent Kinases 4/6 Activity in Pancreatic Ductal Adenocarcinoma(2016-06-06) Franco, Jorge; Brekken, Rolf A.; Burma, Sandeep; Pearson, Gray W.; Knudsen, Erik S.Pancreatic ductal Adenocarcinoma (PDA) is an aggressive and lethal disease that lacks an adequate treatment. Given that patients with PDA only marginally benefit from the current therapies, there is an urgent need to develop more effective approaches that specifically target PDA. Because a significant portion of PDA tumors lose p16 expression and often over-express Cyclin D1, we hypothesized that CDK4/6 activity is deregulated leading to uncontrolled proliferation. Thus, PDA would represent a good candidate for treatment with recently developed CDK4/6 inhibitors. Our study first investigated the sensitivity of PDA cells to CDK4/6 inhibition and found that PDA cells exhibit variable responses to CDK4/6 inhibition, incling models that display significant resistance. Interestingly, these models showed a novel mechanism of resistance to CDK4/6 inhibition. Prior to this study, RB loss was the only mechanism known to circumvent CDK4/6 inhibition. However, herein, we uncovered that CDK4/6 inhibition can lead to aberrant Cyclin E expression, which can compensate for CDK4/6 activity loss and maintain RB in a hyper-phosphorylated state. Subsequent findings demonstrated that this resistance could be blocked by combination therapy with MTOR inhibitors, which prevented aberrant Cyclin E expression and reinforced RB activation. Our second study found that prolonged CDK4/6 inhibition led to an altered metabolic state with an increased in oxidative respiration and glycolysis accompanied by mitochondria accumulation and increase cellular complexity. This heightened metabolic state was mediated by MTOR signaling, which activity was stimulated by amino acid accumulation and an increase in lysosome production in CDK4/6 treated PDA cells. Lastly, we unveiled new combination therapies that targeted the altered metabolism state of CDK4/6 inhibitor treated cells by impinging on antioxidants such as Hemoxygenase 1 (HO-1) and catalase (CAT), whose expression was enhanced post treatment, or by targeting BCL-2/BCL-XL using ABT-737. Taken together our data demonstrate that targeting PDA with CDK4/6 inhibitors can represent an efficacious route for treatment. Activation of MTOR and perhaps other signaling pathways likely contribute to intrinsic and acquired resistance to CDK4/6. These combined data would support the combined use of CDK4/6 with MTOR inhibitors and other agents for the treatment of pancreatic cancer.