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The serine/threonine kinase Akt mediates mitogenic and anti-apoptotic responses that result from activation of multiple signaling cascades. It is considered a key determinant of tumor aggressiveness and is a major target for anticancer drug development. Here, we describe a new reporter molecule whose bioluminescence activity within live cells and in mice can be used to measure Akt activity. Akt activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to activation or inhibition of receptor tyrosine kinase, inhibition of phosphoinositide 3-kinase, or direct inhibition of Akt. The results provide unique insights into the pharmacokinetics and pharmacodynamics of agents that modulate Akt activity, revealing the usefulness of this reporter for rapid dose and schedule optimization in the drug development process., Author(s): Limin Zhang [1]; Kuei C Lee [2]; Mahaveer S Bhojani [1]; Amjad P Khan [1]; Alla Shilman [1]; Eric C Holland [3]; Brian D Ross [2, 4]; Alnawaz Rehemtulla [...]

In an effort to identify a clinical biomarker for lung cancer, we used cDNA microarray and 2D protein analyses to demonstrate that increased Fas-associated death domain (FADD) mRNA and protein were significantly associated with poor survival. Analyses of copy number and sequence of the FADD gene in 24 independent tumors ruled out the existence of an amplified and/or mutated FADD gene in aggressive lung cancers. Immunohistochemistry-based tissue microarray analysis showed that nuclear localization of FADD and elevation of the phosphorylated form of FADD (p-FADD) correlated with poor outcome (P = 0.003). Tumors with increased p-FADD expression showed elevated NF-[kappa]B (P = 0.004) activation, a frequent molecular alteration associated with tumorigenesis and metastasis in a variety of cancers. To provide a link between p-FADD and NF-[kappa]B, cell culture studies demonstrated that overexpression of p-FADD leads to an increase in NF-[kappa]B activity and a decrease in the number of cells in the G2 phase of the cell cycle, compared with cells expressing the nonphosphorylatable form of FADD or the vector control. Furthermore, cDNA microarray analyses of lung tumor samples showed that increased levels of FADD transcripts were significantly correlated with overexpression of cyclins D1 (P < 0.01) and B1 (P < 0.01), genes that are involved in the regulation of cell cycle progression and are inducible by NF-[kappa]B. These studies demonstrate that induction of NF-[kappa]B activity and its effects on cell-cycle progression may represent a molecular basis underlying the aggressive tumor behavior associated with elevated p-FADD expression in lung adenocarcinoma. survival | proliferation | lung cancer

Abstract Recent advances in oncology have lead to identification of a plethora of alterations in signaling pathways that are critical to oncogenesis and propagation of malignancy. Among the biomarkers identified, dysregulated kinases and associated changes in signaling cascade received the lion's share of scientific attention and have been under extensive investigations with goal of targeting them for anti-cancer therapy. Discovery of new drugs is immensely facilitated by molecular imaging technology which enables non-invasive, real time, dynamic imaging and quantification of kinase activity. Here, we review recent development of novel kinase reporters based on conformation dependent complementation of firefly luciferase to monitor kinase activity. Such reporter system provides unique insights into the pharmacokinetics and pharmacodynamics of drugs that modulate kinase signaling and have a huge potential in drug discovery, validation, and drug-target interactions.

Vascular-targeted therapies have shown promise as adjuvant cancer treatment. As these agents undergo clinical evaluation, sensitive imaging biomarkers are needed to assess drug target interaction and treatment response. In this study, dynamic contrast enhanced MRI (DCE-MRI) and diffusion-weighted MRI (DW-MRI) were evaluated for detecting response of intracerebral 9 L gliosarcomas to the antivascular agent VEGF-Trap, a fusion protein designed to bind all forms of Vascular Endothelial Growth Factor-A (VEGF-A) and Placental Growth Factor (PGF). Rats with 9 L tumors were treated twice weekly for two weeks with vehicle or VEGF-Trap. DCE- and DW-MRI were performed one day prior to treatment initiation and one day following each administered dose. Kinetic parameters ( Ktrans, volume transfer constant; kep, efflux rate constant from extravascular/extracellular space to plasma; and vp, blood plasma volume fraction) and the apparent diffusion coefficient (ADC) over the tumor volumes were compared between groups. A significant decrease in kinetic parameters was observed 24 hours following the first dose of VEGF-Trap in treated versus control animals ( p < 0.05) and was accompanied by a decline in ADC values. In addition to the significant hemodynamic effect, VEGF-Trap treated animals exhibited significantly longer tumor doubling times ( p < 0.05) compared to the controls. Histological findings were found to support imaging response metrics. In conclusion, kinetic MRI parameters and change in ADC have been found to serve as sensitive and early biomarkers of VEGF-Trap anti-vascular targeted therapy. Copyright © 2011 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The serine/threonine kinase PKB/Akt is a key mediator of survival and resistance to cancer therapy. Pharmacologic inhibition of Akt and its biologic sequelae may significantly impact the treatment of cancer. The use of molecular imaging technologies has contributed significantly to drug discovery research with an emphasis on drug efficacy, the mechanism of action, and target validation studies. We constructed a genetically engineered hybrid bioluminescent Akt reporter (BAR) molecule that reports on Akt serine/threonine kinase activity. Based on the fact that Akt is recruited to the plasma membrane on activation, we here describe a modified version of this reporter molecule (myristoylated and palmitoylated bioluminescent Akt reporter [MyrPalm-BAR]), which is membrane bound and whose bioluminescence activity can be used to monitor Akt activity at the cell membrane. Using changes in Akt activation status with small molecule inhibitors of Akt, we demonstrated that the membrane-targeted Akt reporter was more sensitive and quantitative. In addition, inhibition of upstream signaling kinases such as epidermal growth factor receptor and phosphatidylinositol 3-kinase activity resulted in changes in Akt activity, that were quantitatively monitored by bioluminescence imaging. Based on these results, we propose that the membrane-associated Akt reporter may be better suited for identification of novel compounds that modulate the Akt pathway by high-throughput screening. [ABSTRACT FROM AUTHOR]

Chemokines and chemokine receptors have been posited to have important roles in several common malignancies, including breast and lung cancer. Here, we demonstrate that CXCR7 (RDC1, CCX-CKR2), recently deorphanized as a chemokine receptor that binds chemokines CXCL11 and CXCL12, can regulate these two common malignancies. Using a combination of overexpression and RNA interference, we establish that CXCR7 promotes growth of tumors formed from breast and lung cancer cells and enhances experimental lung metastases in immunodeficient as well as immunocompetent mouse models of cancer. These effects did not depend on expression of the related receptor CXCR4. Furthermore, immunohistochemistry of primary human tumor tissue demonstrates extensive CXCR7 expression in human breast and lung cancers, where it is highly expressed on a majority of tumor-associated blood vessels and malignant cells but not expressed on normal vasculature. In addition, a critical role for CXCR7 in vascular formation and angiogenesis during development is demonstrated by using morpholino-mediated knockdown of CXCR7 in zebrafish. Taken together, these data suggest that CXCR7 has key functions in promoting tumor development and progression. [ABSTRACT FROM AUTHOR]

The serine/threonine kinase Akt mediates mitogenic and anti-apoptotic responses that result from activation of multiple signaling cascades. It is considered a key determinant of tumor aggressiveness and is a major target for anticancer drug development. Here, we describe a new reporter molecule whose bioluminescence activity within live cells and in mice can be used to measure Akt activity. Akt activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to activation or inhibition of receptor tyrosine kinase, inhibition of phosphoinositide 3-kinase, or direct inhibition of Akt. The results provide unique insights into the pharmacokinetics and pharmacodynamics of agents that modulate Akt activity, revealing the usefulness of this reporter for rapid dose and schedule optimization in the drug development process. [ABSTRACT FROM AUTHOR]

In an effort to identify a clinical biomarker for lung cancer, we used cDNA microarray and 2D protein analyses to demonstrate that increased Fas-associated death domain (FADD) mRNA and protein were significantly associated with poor survival. Analyses of copy number and sequence of the FADD gene in 24 independent tumors ruled out the existence of an amplified and/or mutated FADD gene in aggressive lung cancers. Immunohistochemistry-based tissue microarray analysis showed that nuclear localization of FADD and elevation of the phosphorylated form of FADD (p-FADD) correlated with poor outcome (P = 0.003). Tumors with increased p-FADD expression showed elevated NP-κB (P = 0.004) activation, a frequent molecular alteration associated with tumorigenesis and metastasis in a variety of cancers. To provide a link between p-FADD and NP-κB, cell culture studies demonstrated that overexpression of p-FADD leads to an increase in NF-κB activity and a decrease in the number of cells in the G2 phase of the cell cycle, compared with cells expressing the nonphosphorylatable form of FADD or the vector control. Furthermore, cDNA microarray analyses of lung tumor samples showed that increased levels of FADD transcripts were significantly correlated with overexpression of cyclins D1 (P < 0.01) and B1 (P < 0.01), genes that are involved in the regulation of cell cycle progression and are inducible by NF-κB. These studies demonstrate that induction of NF-κB activity and its effects on cell-cycle progression may represent a molecular basis underlying the aggressive tumor behavior associated with elevated p-FADD expression in lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Pancreatic cancer is a highly lethal disease with a poor prognosis characterized by local and systemic disease progression. Both radiation and chemotherapy play important roles in the management of this disease. However, in order to improve standard therapy many molecularly targeted agents are being developed. Glycogen synthase kinase 3β (GSK3β) participates in a multitude of cellular processes and is a newly proposed therapeutic target in pancreatic cancer. This review will discuss both the oncogenic and tumor suppressor functions of GSK3β in pancreatic cancer with an emphasis on the roles of GSK3β in tumor cell survival and sensitivity to radiation and chemotherapy.

The transcription factor, nuclear factor kappaB (NF-kappaB), plays a central role as a key mediator of cell survival and proliferation, and its activation may confer increased tumor chemoresistance. Curcumin, an orally available naturally occurring compound, has been shown to inhibit NF-kappaB and has a potential role in cancer chemoprevention. We investigated the effects of curcumin on NF-kappaB activity, on cell viability, and as a chemosensitizing agent with 5-fluorouracil (5-FU) or cisplatin (CDDP) in esophageal adenocarcinoma (EAC). Oligonucleotide microarray analysis of 46 cases, consisting of Barrett metaplasia, low-grade dysplasia, high-grade dysplasia and EAC, showed increased expression of NF-kappaB and IkappaB kinase subunits and decreased effector caspase expression in EAC compared with Barrett metaplasia. Stromal expression of both IkappaB and phospho-IkappaB was detected in several EAC samples by tissue microarray analysis. Curcumin alone inhibited NF-kappaB activity and induced apoptosis in both Flo-1 and OE33 EAC cell lines as determined by Western blot analysis, NF-kappaB reporter assays, and Caspase-Glo 3/7 assays. It also increased 5-FU- and CDDP-induced apoptosis in both cell lines. These data suggest that activation of NF-kappaB and inhibition of apoptosis may play a role in the progression from Barrett metaplasia to EAC. In addition, curcumin, a well-known inhibitor of NF-kappaB activity, was shown to increase apoptosis and enhance both 5-FU- and CDDP-mediated chemosensitivity, suggesting that it may have potential application in the therapy of patients with EAC.