Development of an immunohistochemical protein quantification system in conjunction with tissue microarray technology for identifying predictive biomarkers for phosphatidylinositol 3-kinase inhibitors.

The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in human cancers by gain-of-function mutations of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) or dysfunction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Therefore PI3K is thought to be a promising target for cancer therapy. Many agents targeting PI3K have been developed and some of them have been evaluated in clinical trials. In recent years, development of predictive biomarkers as companion diagnostics for molecular targeted drugs has become an important requirement for clinical development; however, no clinically established biomarkers that predict the efficacy of PI3K inhibitors have been found. We previously reported that expression of phosphorylated Akt determined by immunoblot analysis correlated with the antitumor efficacy of a PI3K inhibitor ZSTK474 in vitro and in vivo, suggesting that it might be used as a predictive biomarker. In this study, to evaluate biomarker candidates in in vivo tumor samples, we developed an immunohistochemical protein detection/quantification system in conjunction with the tissue microarray technology using a panel of 24 human tumor xenografts (JFCR24). We have clearly demonstrated that expression levels of phosphorylated v-akt murine thymoma viral oncogene homolog (Akt) and mitogen-activated protein kinase (MAPK) determined by this system significantly correlated with those determined by immunoblot analysis. As expected, PTEN status correlated with expression of phosphorylated Akt but not MAPK. Finally, we confirmed that phosphorylated Akt levels determined using this system correlated with the in vivo efficacy of ZSTK474. The present results indicate that the immunohistochemical protein detection/quantification system could be used to quantify expression of biomarker proteins in xenografted tumor tissues as well as in human tumor specimens to predict drug efficacy in future clinical trials.

Establishment of phosphatidylinositol 3-kinase inhibitor-resistant cancer cell lines and therapeutic strategies for overcoming the resistance.

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.

Correlating phosphatidylinositol 3-kinase inhibitor efficacy with signaling pathway status: in silico and biological evaluations.

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.

Estrogenic and Acetylcholinesterase-Enhancement Activity of a New Isoflavone, 7,2',4'-Trihydroxyisoflavone-4'-O-beta-D-Glucopyranoside from Crotalaria Sessililflora.

A new isoflavone, 7,2',4'-trihydroxyisoflavone-4'-O-beta-D-glucopyranoside has been isolated from the aerial part of Crotalaria sessiliflora. The isoflavone glucoside enhanced the proliferation of the MCF-7 human breast cancer cell line, which possesses estrogen receptor (ER) and responds to estrogen in culture. The estrogenic property of the isoflavone glucoside was blocked by the known ER antagonist tamoxifen, indicating the involvement of the ER. Furthermore, the isoflavone glucoside was found to enhance the acetylcholinesterase (AChE) activity of the rat neuronal cell line PC12 at low concentrations of nerve growth factor (NGF).