OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

HLA-BAT1 alters migration, invasion and pro-inflammatory cytokines in prostate cancer.

Prostate cancer (PCa) accounts for more than 1 in 5 diagnoses and is the second cause of cancer-related deaths in men. Although PCa may be successfully treated, patients may undergo cancer recurrence and there is a need for new biomarkers to improve the prediction of prostate cancer recurrence and improve treatment. Our laboratory demonstrated that HLA-B-associated transcript 1 (BAT1) was differentially expressed in patients with high Gleason scores when compared to low Gleason scores. BAT1 is an anti-inflammatory gene but its role in PCa has not been identified. The objective of this study is to understand the role of BAT1 in prostate cancer. In vitro studies showed that BAT1 down-regulation increased cell migration and invasion. In contrast, BAT1 overexpression decreased cell migration and invasion. RT-PCR analysis showed differential expression of pro-inflammatory cytokines (TNF-α and IL-6) and cell adhesion and migration genes (MMP10, MMP13, and TIMPs) in BAT1 overexpressed cells when compared to BAT1 siRNA cells. Our in vivo studies demonstrated up-regulation of TNF-α, IL-6, and MMP10 in tumors developed from transfected BAT1 shRNA cells when compared to tumors developed from BAT1 cDNA cells. These findings indicate that BAT1 down-regulation modulates TNF-α and IL-6 expression which may lead to the secretion of MMP-10 and inhibition of TIMP2.

Targeting the Platelet-Derived Growth Factor-beta Stimulatory Circuitry to Control Retinoblastoma Seeds.

Purpose: Vitreous seeding remains the primary reason for treatment failure in eyes with retinoblastoma (Rb). Systemic and intra-arterial chemotherapy, each with its own inherent set of complications, have improved salvage rates for eyes with advanced disease, but the location and biology of vitreous seeds present a fundamental challenge in developing treatments with minimal toxicity and risk. The aim of this study was to target the platelet-derived growth factor (PDGF)- PDGF-receptor ß (PDGFRß) signaling pathway and investigate its role in the growth of Rb seeds, apoptotic activity, and invasive potential. Methods: We performed ex vivo analyses on vitreous samples from Rb patients that underwent enucleation and from patient-derived xenografts. These samples were evaluated by quantitative PCR, immunohistochemistry, and ELISA. The effects of disruption of the PDGF-PDGFRß signaling pathway, both by pharmacologic and genomic knockdown approaches, were evaluated in vitro by cell proliferation and apoptotic assays, quantitative PCR analyses, Western blotting, flow cytometry, and imaging flow cytometry. A three-dimensional cell culture system was generated for in-depth study of Rb seeds. Results: Our results demonstrated that PDGFRß signaling is active in the vitreous of Rb patients and patient-derived xenografts, sustaining growth and survival in an AKT-, MDM2-, and NF-κB-dependent manner. The novel three-dimensional cell culture system mimics Rb seeds, as the in vitro generated spheroids have similar morphologic features to Rb seeds and mimicked their natural physiology. Conclusions: Targeting the PDGFRß pathway in vitro reduces Rb cell growth, survival, and invasiveness and could augment current therapies. This represents a novel signaling pathway for potential targeted therapy to further improve ocular survival in advanced Rb.