Purpose: The dual modality of TGFb, both as a potent tumor suppressor and a stimulator of tumor progression, invasion, and metastasis, make it a critical target for therapeutic intervention in human cancers. The ability to carry out real-time, noninvasive imaging of TGFb-activated Smad signaling in live cells and animal models would significantly improve our understanding of the regulation of this unique signaling cascade. To advance these efforts, we developed a highly sensitive molecular imaging tool that repetitively, noninvasively, and dynamically reports on TGFBR1 kinase activity.Experimental Design: The bioluminescent TGFbR1 reporter construct was developed using a split firefly luciferase gene containing a functional sensor of Smad2 phosphorylation, wherein inhibition of TGFb receptor1 kinase activity leads to an increase in reporter signaling. The reporter was stably transfected into mammalian cells and used to image in vivo and in vitro bioluminescent activity as a surrogate for monitoring TGFBR1 kinase activity.Results: The reporter was successfully used to monitor direct and indirect inhibition of TGFb-induced Smad2 and SMAD3 phosphorylation in live cells and tumor xenografts and adapted for high-throughput screening, to identify a role for receptor tyrosine kinase inhibitors as modulators of TGFb signaling.Conclusion: The reporter is a dynamic, noninvasive imaging modality for monitoring TGFb-induced Smad2 signaling in live cells and tumor xenografts. It has immense potential for identifying novel effectors of R-Smad phosphorylation, for validating drug-target interaction, and for studying TGFb signaling in different metastasis models. Clin Cancer Res; 17(23); 7424-39. Ó2011 AACR.
Fas-associated protein with death domain (FADD) was originally reported as a proapoptotic adaptor molecule that mediates receptor-induced apoptosis. Recent studies have revealed a potential role of FADD in NF-κB activation, embryogenesis, and cell cycle regulation and proliferation. Overexpression of FADD and its phosphorylation have been associated with the transformed phenotype in many cancers and is therefore a potential target for therapeutic intervention. In an effort to delineate signaling events that lead to FADD phosphorylation and to identify novel compounds that impinge on this pathway, the authors developed a cell-based reporter for FADD kinase activity. The reporter assay, optimized for a high-throughput screen (HTS), measures bioluminescence in response to modulation of FADD kinase activity in live cells. In addition, the potential use of the reporter cell line in the rapid evaluation of pharmacologic properties of HTS hits in mouse models has been demonstrated.
FADD (Fas-associated protein with death domain) is a cytosolic adapter protein essential for mediating death receptor-induced apoptosis. It has also been implicated in a number of non-apoptotic activities including embryogenesis, cell-cycle progression, cell proliferation, and tumorigenesis. Our recent studies have demonstrated that high levels of phosphorylated FADD in tumor cells correlates with increased activation of the anti-apoptotic transcription factor NF-κB and is a biomarker for aggressive disease and poor clinical outcome. These findings suggest that inhibition of FADD phosphorylation is a viable target for cancer therapy. A high throughput screen using a cell-based assay for monitoring FADD-kinase activity identified NSC 47147 as a small molecule inhibitor of FADD phosphorylation. The compound was evaluated in live cells and mouse tumors for its efficacy as an inhibitor of FADD-kinase activity through the inhibition of CK1α. NSC 47147 was shown to decrease levels of phosphorylated FADD and NF-κB activity such that combination therapy lead to greater induction of apoptosis and enhanced tumor control as compared to either agent alone. The studies described here demonstrate the utility of bioluminescent cell based assays for the identification of active compounds and the validation of drug target interaction in a living subject. In addition, the presented results provide proof of principle studies as to the validity of targeting FADD-kinase activity as a novel cancer therapy strategy.
Fas-Associated protein with Death Domain (FADD) was originally reported as a pro-apoptotic adaptor molecule that mediates receptor induced apoptosis. Recent studies have revealed a potential role of FADD in NF-κB activation, embryogenesis, and cell cycle regulation and proliferation. Over-expression of FADD and its phosphorylation have been associated with the transformed phenotype in many cancers and is therefore a potential target for therapeutic intervention. In an effort to identify novel chemical entities that inhibit FADD phosphorylation, we screened an NCI diversity compound collection using a cell based assay recently developed in our laboratory for the detection of FADD-kinase activity (Khan, 2010). The screen resulted in the identification of NSC 47147 as a potent inhibitor of FADD phosphorylation. This compound was shown to be an inhibitor of CKIα, a previously described FADD kinase, with an IC50 = 1.5 µM. We demonstrate that inhibition of FADD phosphorylation by NSC 47147 results in a decrease in NF-κB activity, thereby potentially sensitizing tumor cells to chemotherapy. In support of this, treatment of lung cancer cells with NSC 47147 and cisplatin in culture as well as in a mouse xenograft model resulted in the synergistic induction of apoptosis and enhanced tumor control compared to either agent alone. These results provide evidence for the inhibition of FADD phosphorylation as a viable target for anti-cancer therapy.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2311. doi:10.1158/1538-7445.AM2011-2311
Induction of cell death in malignant lesions still represents the major goal in the treatment of cancer patients. Imaging of apoptosis would enhance the development of optimal dosing, schedule and combination therapies. Here we used a luciferase biosensor (Promega) and developed a bioluminescent assay system with optimized signal to noise for the detection of cell death in vitro and in living animals. Using D54-MG cells stably expressing the reporter, we demonstrate that TRAIL treatment resulted in a 100–200 fold induction of bioluminescence activity which correlated with cell death as demonstrated by increased cleavage of Caspase-3. Furthermore inhibition of apoptosis using the pan caspase inhibitor Z-VAD-FMK resulted in inhibition of reporter activity. The utility of this cell death reporter was further demonstrated in human glioma xenografted tumors wherein induction of apoptosis was correlated with reporter activation. The high signal to noise and dynamic range of reporter activity provides for a sensitive and quantitative surrogate for evaluation of experimental therapeutics. In addition, the ability to image repeatedly provides a unique opportunity to understand the dynamics of cell death in response to specific drugs or combination therapies. We are also investigating the application of this technology in a high throughput screening of compound and targeted siRNA libraries in diverse cells and in pre-clinical cancer mouse models.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-334. doi:10.1158/1538-7445.AM2011-LB-334
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