Akt sits at the crossroads of numerous signal tansduction pathways essential for human tumorigenesis and activation of the PI3K/Akt signaling is a hallmark of many human cancers. Inhibition of this critical transforming pathway is closely linked with the anti-tumor effects of many targeted therapies and the ability to inhibit Akt signaling in cell culture models has become an important benchmark for the evaluation of targeted anti-cancer drugs and a molecular marker of response and resistance to many therapies. The ability to non-invasively measure PI3K/Akt signaling activity in vivo is currently lacking. Such a modality could allow much better patient selection for therapies, could predict reponse to therapy and could potentially predict relapse on therapy. Such a modality would allow the unprecedented opportunity for dose escalation or reduction in individual patients guided by a readout of tumor PI3K/Akt activity. An imaging modality to monitor Akt activity in vivo is urgently needed. The technology to directly measure intracellular signaling events in tumors in vivo is currently not available. However cell surface probes can potentially be used as reporters of intraceilular signaling as evidenced by the development of annexin V as a probe of early intracellular apoptotic signaling. Indeed the PI3K/Akt signaling pathway regulates endosomal trafficking and membrane transport, including the expression of several membrane metabolic transporters, and regulates gene expression through several transcription factors and it is likely to modulate the cell surface proteome. Based on the hypothesis that tumor cells express certain surface epitopes uniquely in the PI3K/Akt activated state, we seek to develop probes that bind such epitopes and have developed a cell model well suited for this effort. Our cell model consists of engineered MDA-468 breast cancer cells which have highly active Akt signaling due to deletion of PTEN, and in which we can rapidly inactivate Akt signaling by tet-induced expression of PTEN. We propose to develop probes that will bind to the surface of these cells in the Akt-active state, but not the Akt-inactive state. We will subtractively screen a single chain antibody (scFv) phage library to identify probes that will bind selectively to the surface of Akt-activated cells, but not the Akt-inactive cells. Preliminarily identified probes will be further validated in broader cell contexts. Validated probes will be F-18 radiolabelled, and the sensitivity of candidate probes in generating a signal in Akt-activated tumors will be preliminarily assessed by micro-PET studies of mice bearing uninduced MDA-468TR-PTEN xenograft tumors. The specificity of such probes will be studied by microPET studies of the same mice after doxycycline feeding to inactive tumor Akt signaling. If successful, this modality could potentially lead to a ground-breaking predictive clinical monitoring tool. ? ? ?
