The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in human cancers, and several agents targeting this pathway including PI3K/Akt/mammalian target of rapamycin inhibitors have recently entered clinical trials. One question is whether the efficacy of a PI3K pathway inhibitor can be predicted based on the activation status of pathway members. In this study, we examined the mutation, expression, and phosphorylation status of PI3K and Ras pathway members in a panel of 39 pharmacologically well-characterized human cancer cell lines (JFCR39). Additionally, we evaluated the in vitro efficacy of 25 PI3K pathway inhibitors in addition to conventional anticancer drugs, combining these data to construct an integrated database of pathway activation status and drug efficacies (JFCR39-DB). In silico analysis of JFCR39-DB enabled us to evaluate correlations between the status of pathway members and the efficacy of PI3K inhibitors. For example, phospho-Akt and KRAS/BRAF mutations prominently correlated with the efficacy and the inefficacy of PI3K inhibitors, respectively, whereas PIK3CA mutation and PTEN loss did not. These correlations were confirmed in human tumor xenografts in vivo, consistent with their ability to serve as predictive biomarkers. Our findings show that JFCR39-DB is a useful tool to identify predictive biomarkers and to study the molecular pharmacology of the PI3K pathway in cancer. Cancer Res; 70(12); 4982-94. ©2010 AACR.

Cofilin, a 21-kDa actin-binding protein, has a hexapeptide sequence DAIKKK which is identical to the N-terminal portion (residues 2 -7) of tropomyosin. The synthetic heptapeptide, DAIKKKL, corresponding to residues 122 -128 of cofilin, inhibited the binding of cofilin to F-actin in a dose-dependent manner. The heptapeptide cosedimented with F-actin, decreased the fluorescence intensity of pyrene-labeled F-actin, and increased the rate of polymerization of G-actin. The hexapeptides, DIKKKL and DAIKKL, also inhibited the binding of cofilin to F-actin and affected the fluorescence intensity of pyrene-labeled F-actin and the rate of actin polymerization, like the heptapeptide. However, their effects were weaker than those of the heptapeptide. Moreover, the pentapeptide, DIKKL, had little or no effect. These results suggest that the heptapeptide sequence is specific for the interaction with actin and, therefore, may constitute part of the actin-binding domain of cofilin.Cofilin is a ubiquitous actin-modulating protein that binds to both G-actin and F-actin in a 1 : 1 molar ratio of cofilin to actin molecules in vitro [l -41. It shortens the average length of F-actin and increases the steady-state concentration of G-actin to a limited extent at neutral pH [l]. Moreover, cofilin is shown to control actin polymerization in a pH-dependent manner in vitro [5].Recently, we have determined the entire amino acid sequence of cofilin derived from porcine brain by cDNA cloning and sequencing [6]. Cofilin has a hexapeptide DAIKKK (residues 122 -127) identical to the amino-terminal sequence (residues 2 -7) of tropomyosin. This is interesting because cofilin and tropomyosin compete with each other for binding to F-actin [l]. Cofilin could be cross-linked to the aminoterminal region of actin molecule with a zero-length crosslinker which links a carboxyl group to an amino group [7]. Because the amino-terminal portion of the actin molecule is rich in acidic residues, basic amino acid residues of cofilin may be involved in the binding of cofilin to actin [7]. The fact that the binding of cofilin to actin is inhibited at high ionic strength (2, 81 also suggests the importance of electrostatic interactions between cofilin and actin. Thus, the DAIKKK sequence, which contains three consecutive basic residues, is a possible candidate for an actin-binding site in the cofilin sequence.In this study, we synthesized a DAIKKKL heptapeptide corresponding to residues 122 -128 of cofilin, and examined its effect on the binding of cofilin to actin and its direct interaction with actin. The actions of other synthetic peptides, DIKKKL, DAIKKL and DIKKL, were also examined.Correspondence to E. Nishida, Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo-shi, Tokyo-to, Japan 113 MATERIALS AND METHODS ProteinsCofilin was purified from porcine brain by the method described previously (81. Actin was prepared from rabbit skeletal muscle according to the method of Spudich and Watt [9], and furth...

Acquired resistance is a major obstacle for conventional cancer chemotherapy, and also for some of the targeted therapies approved to date. Long‐term treatment using protein tyrosine kinase inhibitors (TKIs), such as gefitinib and imatinib, gives rise to resistant cancer cells carrying a drug‐resistant gatekeeper mutation in the kinase domain of the respective target genes, EGFR and BCR–ABL. As for the phosphatidylinositol 3‐kinase inhibitors (PI3Kis), little is known about their acquired resistance, although some are undergoing clinical trials. To address this issue, we exposed 11 human cancer cell lines to ZSTK474, a PI3Ki we developed previously, for a period of more than 1 year in vitro. Consequently, we established ZSTK474‐resistant cells from four of the 11 cancer cell lines tested. The acquired resistance was not only to ZSTK474 but also to other PI3Kis. None of the PI3Ki‐resistant cells, however, contained any mutation in the kinase domain of the PIK3CA gene. Instead, we found that insulin‐like growth factor 1 receptor (IGF1R) was overexpressed in all four resistant cells. Interestingly, targeted knockdown of IGF1R expression using specific siRNAs or inhibition of IGF1R using IGF1R‐TKIs reversed the acquired PI3Ki resistance. These results suggest that long‐term treatment with PI3Kis may cause acquired resistance, and targeting IGF1R is a promising strategy to overcome the resistance.

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