Inhibition of AKT final results in upregulation of RTKs in vitro and in vivo We and other people have previously reported that inhibition of PI3K/AKT/mTOR induces compensatory expression and activation of many RTKs. In an effort to iden tify inhibitors that can be rationally combined together with the AKT antagonist in hormone independent breast cancer, we examined the effects of AZD5363 on the set of thera peutically targetable RTKs. Treatment with AZD5363 upregulated mRNA levels of many RTKs, with InsR, HER3 and IGF IR remaining the major hits across all four LTED lines. FGFR 2 4 mRNAs have been also induced upon therapy with AZD5363. Inhibition of AKT resulted in upregulation of complete and phosphory lated HER3 in three of the 4 LTED lines, too as Y416 P Src protein amounts. Remedy with two ?M AZD5363 upregulated InsR protein one.
4 fold in MCF 7/LTED cells and 5. 7 fold in MDA 361/LTED cells. Treatment using the Src kinase inhibitor dasatinib great post to read decreased AZD5363 induced upregulation of phosphorylated HER3 in MCF 7/LTED cells, also as drastically enhanced the development inhibitory results of AZD5363. Even so, treatment using the Src inhibitor AZD0530 was ineffective. Pre treatment together with the IGF IR/InsR dual TKI AEW541 or BKM120 abrogated the AZD5363 induced enhance in P Src, suggesting the enhance in active Src was on account of activation of IGF IR/ InsR and PI3K. We following assessed the results of AZD5363 on a wider panel of RTKs. Following inhibition of AKT in MCF 7/ LTED, ZR75 1/LTED and MDA 361/LTED cells, phos pho RTK array evaluation unveiled improved phosphorylation of numerous RTKs, which include InsR, IGF IR, HER3, EGFR, HER2, HER4, Dtk, VEGFR1 and FGFR2 4.
To validate these findings in vivo, we treated ovariectomized mice bearing MCF seven xenografts with AZD5363 for 1 or 3 days. Inhibition of AKT upregulated the tumor ranges of P InsR/IGF IR, InsR, P HER3, HER3, P HER2, HER2, the FGFR substrate this article P FRS2 and FGFR2 proteins. More, treat ment with AZD5363 for a single to 3 days also enhanced tumor amounts of InsR, IGF IR and FGFR one 4 mRNAs. Inhibition of IGF IR/InsR or PI3K abrogates AZD5363 induced AKT membrane localization and phosphorylation We speculated that upregulation of activated InsR/IGF IR was keeping PI3K activity and PIP3 formation to counteract the inhibition of AKT and, thus, limit the action of AZD5363.
To test this likelihood, we transfected MCF 7/LTED cells which has a fusion protein comprised from the AKT PH domain fused to the amino terminus of GFP. PIP3 binding towards the PH domain really should lead to translocation of the fusion protein to the plasma mem brane. AKT PH GFP was largely cytoplasmic in control cells, whereas therapy with exogenous IGF I induced its translocation for the membrane. Treatment with AZD5363 also induced marked translocation of AKT PH GFP to your membrane, suggestive of increased PIP3 production and, as a result, AKT phosphorylation on the T308 PDK one web-site.