Aurora B is a mitotic checkpoint kinase that plays a pivotal role in the cell cycle, ensuring correct chromosome segregation and normal progression through mitosis. Aurora B is overexpressed in many types of human cancers, which has made it an attractive target for cancer therapies. Tumor suppressor p53 is a genome guardian and important negative regulator of the cell cycle. Whether Aurora B and p53 are coordinately regulated during the cell cycle is not known. We report that Aurora B directly interacts with p53 at different subcellular localizations and during different phases of the cell cycle (for instance, at the nucleus in interphase and the centromeres in prometaphase of mitosis). We show that Aurora B phosphorylates p53 at S183, T211, and S215 to accelerate the degradation of p53 through the polyubiquitination–proteasome pathway, thus functionally suppressing the expression of p53 target genes involved in cell cycle inhibition and apoptosis (e.g., p21 and PUMA). Pharmacologic inhibition of Aurora B in cancer cells with WT p53 increased p53 protein level and expression of p53 target genes to inhibit tumor growth. Together, these results define a mechanism of p53 inactivation during the cell cycle and imply that oncogenic hyperactivation or overexpression of Aurora B may compromise the tumor suppressor function of p53. We have elucidated the antineoplastic mechanism for Aurora B kinase inhibitors in cancer cells with WT p53.

PURPOSE Survival of patients with osteosarcoma lung metastases has not improved in 20 years. We evaluated the efficacy of combining natural killer [NK] cells with aerosol interleukin-2 [IL-2] to achieve organ-specific NK cell migration and expansion in the metastatic organ, and to decrease toxicity associated with systemic IL-2. EXPERIMENTAL DESIGN Five human osteosarcoma cell lines and 103 patient samples (47 primary and 56 metastatic) were analyzed for NKG2D ligand [NKG2DL] expression. Therapeutic efficacy of aerosol IL-2 + NK cells was evaluated in vivo compared with aerosol IL-2 alone and NK cells without aerosol IL-2. RESULTS Osteosarcoma cell lines and patient samples expressed various levels of NKG2DL. NK-mediated killing was NKG2DL-dependent and correlated with expression levels. Aerosol IL-2 increased NK cell numbers in the lung and within metastatic nodules but not in other organs. Therapeutic efficacy, as judged by tumor number, size, and quantification of apoptosis, was also increased compared with NK cells or aerosol IL-2 alone. There were no IL-2-associated systemic toxicities. CONCLUSION Aerosol IL-2 augmented the efficacy of NK cell therapy against osteosarcoma lung metastasis, without inducing systemic toxicity. Our data suggest that lung-targeted IL-2 delivery circumvents toxicities induced by systemic administration. Combining aerosol IL-2 with NK cell infusions, may be a potential new therapeutic approach for patients with osteosarcoma lung metastasis.

The COP9 signalosome subunit 6 (CSN6), which is involved in ubiquitin-mediated protein degradation, is overexpressed in many types of cancer. CSN6 is critical in causing p53 degradation and malignancy, but its target in cell cycle progression is not fully characterized. Constitutive photomorphogenic 1 (COP1) is an E3 ubiquitin ligase associating with COP9 signalosome to regulate important target proteins for cell growth. p27 is a critical G1 CDK inhibitor involved in cell cycle regulation, but its upstream regulators are not fully characterized. Here, we show that the CSN6-COP1 link is regulating p27(Kip1) stability, and that COP1 is a negative regulator of p27(Kip1). Ectopic expression of CSN6 can decrease the expression of p27(Kip1), while CSN6 knockdown leads to p27(Kip1) stabilization. Mechanistic studies show that CSN6 interacts with p27(Kip1) and facilitates ubiquitin-mediated degradation of p27(Kip1). CSN6-mediated p27 degradation depends on the nuclear export of p27(Kip1), which is regulated through COP1 nuclear exporting signal. COP1 overexpression leads to the cytoplasmic distribution of p27, thereby accelerating p27 degradation. Importantly, the negative impact of COP1 on p27 stability contributes to elevating expression of genes that are suppressed through p27 mediation. Kaplan-Meier analysis of tumor samples demonstrates that high COP1 expression was associated with poor overall survival. These data suggest that tumors with CSN6/COP1 deregulation may have growth advantage by regulating p27 degradation and subsequent impact on p27 targeted genes.

Background We have previously shown that aerosol interleukin-2 [IL-2] increased the number of intravenously injected human natural killer [NK] cells in the lungs. In this study we investigated whether this increase was secondary to NK cell proliferation and determined the site of the proliferation. Materials and Methods Nude mice with osteosarcoma lung metastases were injected with NK cells and treated with aerosol IL-2 or aerosol PBS. BrdU was injected prior to euthanasia to identify proliferating NK cells. The percentage of proliferating NK cells in the lung, bone marrow, spleen and liver was determined using flow cytometry. Survival studies for mice with osteosarcoma lung metastasis treated with aerosol PBS, aerosol IL-2 alone, aerosol PBS plus NK cells and aerosol IL-2 plus NK cells were also performed. Results Treatment with aerosol IL-2 induced the proliferation of injected NK cells in the lung. Aerosol IL-2 did not increase the proliferation of NK cells in the spleen and liver. Treatment with aerosol IL-2 and aerosol IL-2 plus NK cells increased the overall survival of mice with osteosarcoma lung metastasis. Conclusion Aerosol IL-2 increases lung NK cell numbers by stimulating local NK cell proliferation. Aerosol IL-2's effect on NK cell proliferation is organ specific, which makes it ideal for the specific targeting of lung metastasis. Aerosol IL-2 plus NK cell therapy induced metastatic regression and increased overall survival demonstrating the potential of this therapeutic approach for patients with osteosarcoma.