Class III phosphoinositide 3-kinase in melanoma

The first isoform of phosphoinositide 3-kinases (PI3K) had been found in Saccharomyces cerevisiae when screening for mutants not exhibiting normal vacuolar protein sorting (Vps), Vps34. Class III PI3K/Vps34 has long been worked on in regards to its role in endosomal sorting and autophagy, a process allowing cells to survive nutrient-deprived conditions. Most research groups have investigated the functions of Vps34 in the yeast model system Saccharomyces cerevisiae. Newer publications now use mammalian cell lines, Caenorhabditis elegans or Drosophila melanogaster, deciphering interesting differences between the various species in regards to Vps34 characteristics. Our cancer model system, melanoma tumors, are known to be very aggressive and their treatment difficult, due to mutations leading to drug resistance. Autophagy and whether its induction would be beneficial or not for cancer patients, has been the topic of discussions in the field lately.
In this work, we investigated the role of class III PI3K by two different methods, a pharmacological and a genetic approach. We started with natural compound screenings on hVps34 in genetically modified yeast systems. The pure fraction of Citrus medica extracts giving best results turned out to be limettin. Limettin inhibited hVps34 in both our in vivo yeast system and in in vitro kinase assays using the immunoprecipitated enzyme from HEK293 cells. Our candidate inhibitor seemed very specific for the human isoform, but still required quite high concentrations in the assays performed. Further chemical designing and eventual fitting to the hVps34 ATP binding pocket would be necessary to render this molecule into one of the first specific class III PI3K inhibitors.
In addition to pharmacological approaches, class III PI3K state-of-the-art genetic knockdown experiments were done in melanoma cell lines in order to characterize this isoform’s role in melanoma more specifically. Vps34 is not essential in yeast, but leads to serious temperature sensitivity phenotypes. In one melanoma cell line (A375) tested, knockdown had similar as but milder effects than known in other cancer types. Two others though (A2058 and 1205lu), did not tolerate the longterm loss of class III PI3K. We suppose that the importance of hVps34 depends on the genetic background of cell types. Further studies are required to define precisely which effectors determine the intolerance described i.e. which melanoma types could be targeted by inhibition of class III PI3K.