We recently employed a functional drug screening
We recently employed a functional drug screening approach to identify mithramycin as an inhibitor of EWS–FLI1. In our study, we employed a stepwise approach involving a luciferase primary screen and a novel multiplex PCR screen to evaluate over 50,000 compounds to identify mithramycin as an inhibitor of EWS–FLI1 (Grohar, Woldemichael et al., 2011). We showed that mithramycin does not interfere with EWS–FLI1 expression and instead works downstream at the promoter level to block activity, effectively reversing two independent gene signatures of EWS–FLI1 and well-characterized downstream targets such as ID2 and NR0B1 both in vitro and in vivo. Work is ongoing to establish the mechanism of transcriptional interference with EWS–FLI1. It is known that mithramycin blocks the binding of transcription factors to DNA by binding and distorting the minor groove (Banville et al., 1990, Sastry and Patel, 1993, Aich and Dasgupta, 1995). It is established that mithramycin inhibits the SP1 transcription factor, but it is not known what additional transcription factors are specifically blocked by the drug (Ray et al., 1989, Blume et al., 1991, Chatterjee et al., 2001, Remsing et al., 2003, Sleiman et al., 2011, Zhang et al., 2012). Nevertheless, the drug suppresses growth of two different ES xenografts including a marked regression of the TC32 xenograft. Finally, after identifying the compound, we found precedence of mithramycin use in Ewing sarcoma with two CRs reported out of 5 patients treated in the 1960s (Kofman et al., 1973).
An alternative approach to functional drug screening is to characterize critical mediators of EWS–FLI1 activity and screen for compounds that interfere with this interaction. Phage display was used to identify an interaction between EWS–FLI1 and RNA helicase A. This interaction was shown to enhance the activity of the ID2 promoter in Ewing sarcoma Deoxynivalenol where and facilitate soft agar colony formation in Mouse embryonic fibroblasts transformed with EWS–FLI1 (Toretsky et al., 2006). Subsequent to this study, the interaction was further defined and an ex vivo approach was used to identify a small molecule that disrupts this protein–protein interaction to interfere with ES growth in vitro and in vivo (Erkizan et al., 2009). Work is ongoing to develop a drug directed against RNA helicase A in the clinic.
Finally, a more recent screen evaluated the expression of 5 targets of EWS–FLI1 utilizing standard qPCR to screen a library of small molecules to identify the kinase inhibitor, midostaurin as an inhibitor of EWS–FLI1 (Boro et al., 2012). This compound reversed expression of these targets and induced apoptosis of Ewing sarcoma cells both in vitro and in vivo using two different xenograft studies. It is notable that this study also identified camptothecin and doxorubicin as both active agents in the clinic. Doxorubicin as a mainstay of Ewing sarcoma therapy, was previously independently shown to reverse a gene signature of EWS–FLI1 (Stegmaier et al., 2007). Therefore, the contribution of this activity to the mechanism of these drugs in Ewing sarcoma patients is not clear.
Conclusions Finally, continued systematic clinical investigation that integrates active agents such as the camptothecins into Ewing sarcoma therapy will likely lead to improved outcomes or salvage regimens. These methods will undoubtedly evolve and integrate preclinical investigations that identify predictors of sensitivity such as SLFN11 (Barretina et al., 2012). These investigations will be important to the realization of the success of all these agents and hopefully improve survival for patients with Ewing sarcoma.
Introduction Cancer cells survive and proliferate in competition with somatic cells and according to the physical and biological properties of their microenvironment . During cancer progression, as the tumor mass increases in size, neoplastic cells outgrow their blood supply and lack of adequate access to oxygen and nutrients. It is well documented that tumors induce a program of adaptive responses to thrive under hypoxic conditions by switching their metabolism to upregulated glycolysis and by releasing pro-angiogenic factors . However, according to Otto Warburg\'s findings , tumor cells may continue to metabolize carbon by the glycolytic pathway even under adequate oxygen conditions. Therefore, regardless of hypoxia , aerobic glycolysis is a constant feature in cancer development, as suggested by the high levels of glucose consumption detected by positron emission tomography in most malignancies . The high, constant level of glycolytic activity of tumor cells leads to an increased production of lactic acid that decreases the pH of the extracellular microenvironment. Previous authors have demonstrated that the extracellular pH (pHe) of different tumors is in the range of 5.7–7.3 , , , whereas the pHe level of normal tissues is significantly more alkaline (7.2–7.5). The existence of an acidic intracellular pH (pHi) is also indicated in vivo by magnetic resonance spectroscopy . To maintain pH homeostasis and escape apoptosis induced by an increase in proton concentration in the cytosol, cancer cells increase the activity and/or expression of several pH regulators, resulting in the alkalinization of pHi and acidification of pHe . Indeed, an increased expression and activity of the transmembrane vacuolar (H+)-ATPase (V-ATPase) is a constant feature of several tumor types, and its inhibition has been suggested as a promising therapeutic target , , , . V-ATPase is an ATP-driven proton pump that acidifies the intracellular compartment and transports protons across the plasma membranes, both in physiological processes and in human diseases . V-ATPase is a large multisubunit complex composed of a peripheral domain (V1), responsible for hydrolysis of ATP, and an integral domain (V0) that carries out proton transport . In sarcoma cells, the survival mechanism under acidic conditions and the activity of V-ATPase are still entirely unexplored.