osu-03012 and Breast-Neoplasms

Lapatinib is characterized as an ErbB1/ErbB2 dual inhibitor and has recently been approved for the treatment of metastatic breast cancer. In this study, we examined mechanisms associated with enhancing the activity of lapatinib via combination with other therapies.. In the present studies, estrogen receptor (ER) positive and ER negative breast cancer cells were genetically manipulated to up- or downregulate eIF2-alpha, its phospho-mutant, Nck1, or Nck2, then treated with OSU-03012, lapatinib or the combination and assayed for cytotoxicity/cytostaticity using clonogenic assays.. Treatment of breast cancer cell lines with lapatinib and OSU-03012 (a small molecule derivative of the Cox-2 inhibitor celecoxib) induced synergistic cytotoxic/cytostatic effects. This combination therapy corresponded to an increase in the phosphorylation of eIF2-α at serine⁵¹ and a decrease in Nck1 expression. Ectopic expression of phospho-mutant eIF2-α (Ser⁵¹Ala) or downregulation of eIF2-α in addition to downregulation of the eIF2-α kinase PERK inhibited the synergistic and cytotoxic effects. Furthermore, ectopic expression of Nck1, but not Nck2 abolished the decrease in cell viability observed in combination-treated cells. Downregulation of Nck1 failed to "rescue" the ablation of the cytotoxic/cytostatic effects by the phospho-mutant of eIF2-α (Ser⁵¹Ala) demonstrating that Nck1 downregulation is upstream of eIF2-α phosphorylation in the anti-survival pathway activated by lapatinib and OSU-03012 treatment. Finally, co-immunoprecipitation assays indicated that eIF2-α dissociates from the Nck1/PP1 complex after OSU-03012 and lapatinib co-treatment.. These data indicate that OSU-03012 and lapatinib co-treatment is an effective combination therapy, which functions to enhance cell killing through the Nck1/eIF2 complex. Hence, this complex is a novel target for the treatment of metastatic breast cancer.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Immunoprecipitation; Lapatinib; Oncogene Proteins; Pyrazoles; Quinazolines; Signal Transduction; Sulfonamides; Transfection

3-phosphoinositide-dependent protein kinase 1 (PDK1) is the pivotal element of the phosphatidylinositol 3 kinase (PI3K) signaling pathway because it phosphorylates Akt/PKB through interactions with phosphatidylinositol 3,4,5 phosphate. Recent data indicate that PDK1 is overexpressed in many breast carcinomas and that alterations of PDK1 are critical in the context of oncogenic PI3K activation. However, the role of PDK1 in tumor progression is still controversial. Here, we show that PDK1 is required for anchorage-independent and xenograft growth of breast cancer cells harboring either PI3KCA or KRAS mutations. In fact, PDK1 silencing leads to increased anoikis, reduced soft agar growth, and pronounced apoptosis inside tumors. Interestingly, these phenotypes are reverted by PDK1 wild-type but not kinase-dead mutant, suggesting a relevant role of PDK1 kinase activity, even if PDK1 is not relevant for Akt activation here. Indeed, the expression of constitutively active forms of Akt in PDK1 knockdown cells is unable to rescue the anchorage-independent growth. In addition, Akt down-regulation and pharmacological inhibition do not inhibit the effects of PDK1 overexpression. In summary, these results suggest that PDK1 may contribute to breast cancer, even in the absence of PI3K oncogenic mutations and through both Akt-dependent and Akt-independent mechanisms.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Animals; Anoikis; Breast Neoplasms; Cell Proliferation; Cell Survival; Chromones; Female; Humans; Mice; Mice, Nude; Morpholines; Mutation; Nuclear Proteins; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Pyrazoles; ras Proteins; RNA Interference; RNA, Small Interfering; Signal Transduction; Sulfonamides; Transcription Factors; Transplantation, Heterologous

Tamoxifen is a mainstay in the treatment of estrogen receptor (ER)-positive breast cancer patients. Although the efficacy of tamoxifen has been attributed to induction of tumor cell growth arrest and apoptosis by inhibition of ER signaling, recent evidence indicates that tamoxifen possesses ER-independent antitumor activities. Here, we use OSU-03012, a small-molecule inhibitor of phosphoinositide-dependent protein kinase-1 (PDK-1) to address the hypothesis that PDK-1/Akt signaling represents a therapeutically relevant target to sensitize ER-negative breast cancer to tamoxifen. OSU-03012 sensitized both ER-positive MCF-7 and ER-negative MDA-MB-231 cells to the antiproliferative effects of tamoxifen in an ER-independent manner. Flow cytometric analysis of phosphatidylserine externalization revealed that this augmented suppression of cell viability was attributable to a marked enhancement of tamoxifen-induced apoptosis by OSU-03012. Mechanistically, this OSU-03012-mediated sensitization was associated with suppression of a transient tamoxifen-induced elevation of Akt phosphorylation and enhanced modulation of the functional status of multiple Akt downstream effectors, including FOXO3a, GSK3alpha/beta, and p27. The growth of established MDA-MB-231 tumor xenografts was suppressed by 50% after oral treatment with the combination of tamoxifen (60 mg/kg) and OSU-03012 (100 mg/kg), whereas OSU-03012 and tamoxifen alone suppressed growth by 30% and 0%, respectively. These findings indicate that the inhibition of PDK-1/Akt signaling to sensitize ER-negative breast cancer cells to the ER-independent antitumor activities of tamoxifen represents a feasible approach to extending the use of tamoxifen to a broader population of breast cancer patients. Considering the urgent need for novel therapeutic strategies for ER-negative breast cancer patients, this combinatorial approach is worthy of continued investigation.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Animals; Antineoplastic Agents, Hormonal; Apoptosis; Breast Neoplasms; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Drug Synergism; Estrogen Receptor alpha; Female; Flow Cytometry; Forkhead Box Protein O3; Forkhead Transcription Factors; Humans; Immunoblotting; Mice; Mice, Nude; Ovariectomy; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyrazoles; Signal Transduction; Sulfonamides; Tamoxifen; Tumor Cells, Cultured

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 phosphoinositide-dependent 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 -1 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 Array-Scan 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.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Line, Transformed; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Inhibitors; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, SCID; Neoplasm Transplantation; Precipitin Tests; Protein Serine-Threonine Kinases; Pyrazoles; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; RNA, Messenger; RNA, Small Interfering; Sulfonamides; Y-Box-Binding Protein 1

Although trastuzumab has been successfully used in patients with HER2-overexpressing metastatic breast cancer, resistance is a common problem that ultimately culminates in treatment failure. In light of the importance of Akt signaling in trastuzumab's antitumor action, we hypothesized that concurrent inhibition of Akt could enhance trastuzumab sensitivity and moreover reverse the resistant phenotype in HER2-positive breast cancer cells. Based on our finding that celecoxib mediates antitumor effects through the inhibition of phosphoinositide-dependent kinase-1 (PDK-1)/Akt signaling independently of cyclooxygenase-2 (COX-2), we used celecoxib as a scaffold to develop a COX-2-inactive PDK-1 inhibitor, 2-amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012). Here, we investigated the effect of OSU-03012 on trastuzumab-mediated apoptosis in four breast cancer cell lines with different HER2 expression and trastuzumab-resistance status, including MDA-MB-231, BT474, SKBR3, and insulin-like growth factor-I receptor-overexpressing SKBR3 (SKBR3/IGF-IR). Effects of trastuzumab and OSU-03012, individually or in combination, on cell viability and changes in pertinent biomarkers including HER2 expression, phosphorylation of Akt, p27(kip1), and the PDK-1 substrate p70(S6K) were assessed. OSU-03012 alone was able to trigger apoptosis in all cell lines with equal potency (IC(50) = 3-4 microM), suggesting no cross-resistance with trastuzumab. Medium dose-effect analysis indicates that OSU-03012 potentiated trastuzumab's antiproliferative effect in HER2-positive cells, especially in SKBR3/IGF-IR cells, through the down-regulation of PDK-1/Akt signaling. This synergy, however, was not observed in HER2-negative MDA-MB-231 cells. This combination treatment represents a novel strategy to increase the efficacy of trastuzumab and to overcome trastuzumab resistance in the treatment of HER2-positive breast cancer.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Celecoxib; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Gene Expression; Humans; Phenotype; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyrazoles; Receptor, ErbB-2; Receptor, IGF Type 1; Sulfonamides; Trastuzumab; Up-Regulation