The Phosphoinositide-Dependent Kinase-1 Inhibitor 2-Amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012) Prevents Y-Box Binding Protein-1 from Inducing Epidermal Growth Factor Receptor

Abstract

The epidermal growth factor receptor (EGFR) is integral to basal-like and human epidermal growth factor receptor-2 (Her-2)-overexpressing breast cancers. Such tumors are associated with poor prognosis, the majority of which express high levels of EGFR. We reported that EGFR expression is induced by the oncogenic transcription factor Y-box binding protein-1 (YB-1) that occurs in a manner dependent on phosphorylation by Akt. Herein, we questioned whether blocking Akt with 2-amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012), a phosphoinositidedependent protein kinase-1 (PDK-1) small-molecule inhibitor, could prevent YB-1 from binding to the EGFR promoter. MDA-MB-468 and SUM 149 are basal-like breast cancer (BLBC) cells that were used for our studies because they express high levels of activated PDK-1, YB-1, and EGFR compared with the immortalized breast epithelial cell line 184htrt. In these cell lines, YB-1 preferentially bound to the Ϫ1 kilobase of the EGFR promoter, whereas this did not occur in the 184htrt cells based on chromatin immunoprecipitation. When the cells were exposed to OSU-03012 for 6 h, YB-1/EGFR promoter binding was significantly attenuated. To further confirm this observation, gel-shift assays showed that the drug inhibits YB-1/EGFR promoter binding. The inhibitory effect of OSU-03012 on EGFR was also observed at the mRNA and protein levels. OSU-03012 ultimately inhibited the growth of BLBC in monolayer and soft agar coordinate with the induction of apoptosis using an ArrayScan VTI high-content screening system. Furthermore, OSU-03012 inhibited the expression of EGFR by 48% in tumor xenografts derived from MDA-MB-435/Her-2 cells. This correlated with loss of YB-1 binding to the EGFR promoter. Hence, we find that OSU-03012 inhibits YB-1 resulting in a loss of EGFR expression in vitro and in vivo.Breast tumors express characteristic molecular markers that allow for the design and development of targeted drug therapies to control the malignant, deregulated pathways, leaving normal cell systems unscathed. There are five subtypes of breast cancer: luminal A, luminal B, normal-like, This research was supported by grants through the Canadian Breast Cancer Research Alliance, the National Cancer Institute of Canada, and Rethink Breast Cancer (to S.E.D.). Additional funds were provided by SFB542 (to P.R.M.).Article, publication date, and citation information can be found at

PERK-dependent regulation of HSP70 expression and the regulation of autophagy

“…4 OSU-03012 has also been shown to interact in a synergistic fashion with BCR-ABL inhibitors and with the ERBB2 inhibitor Herceptin to suppress tumor cell viability and to kill in a manner that is in many cell types at least partially caspase-independent. [6][7][8][9] In our previous studies, we also noted that inhibition of either MEK1/2 or PI3K enhances the toxicity of OSU-03012 in glioma, colon cancer and transformed rodent fibroblast cell types. 5 However, while 7 Costas Koumenis, 6 Martin Graf, 4 Ching-Shih Chen, 5 OSU-03012 can suppress PDK-1 function and AKT activity, other data have also strongly argued that OSU-03012 toxicity, and its radiosensitizing effects, could not be attributed to suppression of AKT signaling in comparison to siRNA-mediated knock down of PDK-1 protein levels or dominant negative AKT expression.…”

Phosphorylated Insulin-Like Growth Factor-I/Insulin Receptor Is Present in All Breast Cancer Subtypes and Is Related to Poor Survival

“…HTRY cells were immortalized human breast epithelial cells developed from HMEC cells at Wake Forest University, USA (34), and were grown in the same medium as SUM149. Human telomerase-immortalized breast epithelial (184htrt) cells were grown as previously described (35). All cell lines were grown at 37jC with 5% CO 2 .…”

Targeting YB-1 in HER-2 Overexpressing Breast Cancer Cells Induces Apoptosis via the mTOR/STAT3 Pathway and Suppresses Tumor Growth in Mice

“…Annexin V was stained following the manufacturer's protocol (Promega) and the cells were analyzed on a FACSCalibur (BD). Analysis of chromatin condensation, propidium iodidie uptake, and P-H2AX S139 were performed as previously described (7). For propidium iodide staining, the cells were collected after being treated for 4 d with siRNA to YB-1, washed, stained with 30 Ag/mL of propidium iodide (Sigma), and suspended in 500 AL of 1% fetal bovine serum-containing PBS before being analyzed on a FACSCalibur (BD).…”

“…Each of the cell lines were plated at a density of 1,000 cells per 96-well, transfected with siYB-1#2 (5 nmol/L), and a YB-1 knockdown >75% detected by immunofluorescence after 48 h. Subsequently, tumor cell growth was assessed after 72 h by Hoechst staining as previously describing using the ArrayScan VTI (Cellomics; ref. 7). For the siRNA transfections, where changes in signal transduction were monitored, the cells (3 Â 10 5 per 6-well dish to 3.5 Â 10 5 per 6-well dish) were transfected with 5 nmol/L of control or siRNA oligonucleotides according to the manufacturer's protocol.…”