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1.  Molecular Imaging of TGFβ-induced Smad2/3 phosphorylation reveals a role for receptor tyrosine kinases in modulating TGFβ signaling 
The dual modality of TGFβ, 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 perform real-time, noninvasive imaging of TGFβ-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, non-invasively and dynamically reports on TGFBR1 kinase activity.
Experimental Design
The bioluminescent TGFβR1 reporter construct was developed using a split firefly luciferase gene containing a functional sensor of Smad2 phosphorylation wherein inhibition of TGFβ 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.
The reporter was successfully used to monitor direct and indirect inhibitors of TGFβ-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 TGFβ signaling.
The reporter is a dynamic, non-invasive imaging modality for monitoring TGFβ-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 TGFβ signaling in different metastasis models.
PMCID: PMC3229686  PMID: 21948232
bioluminescence; kinase activity; non-invasive molecular imaging; receptor-regulated Smads; TGFβ
2.  A Novel Kinase Inhibitor of FADD Phosphorylation Chemosensitizes through the Inhibition of NF-κB 
Molecular cancer therapeutics  2011;10(10):1807-1817.
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.
PMCID: PMC3191281  PMID: 21859840
FADD; phosphorylation; non-invasive molecular imaging; NF-κB; chemotherapy
3.  High Throughput Molecular Imaging for the Identification of FADD Kinase Inhibitors 
Journal of biomolecular screening  2010;15(9):1063-1070.
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 delineate signaling events that lead to FADD phosphorylation and to identify novel compounds that impinge on this pathway, we 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.
PMCID: PMC3108567  PMID: 20855560
FADD; phosphorylation; non-invasive molecular imaging; bioluminescence; kinase activity

Results 1-3 (3)