Characterization of p27 in Osteosarcoma
Date
2019
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Osteosarcoma is an aggressive bone tumor that primarily affects children and young adults. Patients are treated with high-dose chemotherapy and surgery. However, when metastasis or recurrence occurs it becomes incurable. Osteosarcoma tumors are heterogeneous and pose challenges for selecting targets for new treatments. Biomarkers are needed to screen tumors, and help in the treatment of the disease to better predict patient outcomes. Based on previous studies, our lab focuses on the p27/kip1 (p27) protein as a potential biomarker for osteosarcoma. ☐ p27 is a tumor suppressor that regulates the cell cycle. In cancer cells, p27 localizes to the cytoplasm and interacts with proteins that promote invasion and migration. Therefore, it can also function as an oncoprotein. Osteosarcoma cells use this oncogenic activity to promote proliferation and resistance to therapies. However, a more comprehensive understanding of the role of p27 in osteosarcoma is needed. I hypothesize that nuclear p27 acts as a tumor suppressor, while cytoplasmic p27 acts as an oncoprotein that decreases the sensitivity of osteosarcoma cells to drug treatment. ☐ In this study, I examined how human osteosarcoma cells rely on p27 to evade drug treatments, and allow cells to proliferate in the presence of drugs. I evaluated the activity of the cyclin-dependent kinase 4/6 inhibitor (CDK4/6) palbociclib combined with gemcitabine on the human osteosarcoma cell lines, HOS and 143B. I found that cell viability was reduced in response to gemcitabine and palbociclib given as single agents. When these two drugs were combined, the effect of administering gemcitabine before palbociclib, further reduced cell viability and the cells were sensitive to lower concentrations of palbociclib. ☐ Further studies showed the effect of drug treatment on osteosarcoma cells was dependent on p27 expression. Using immunoblot analysis, I showed a correlation between p27 protein expression and cell viability. Gemcitabine decreased p27 protein levels that correlated with decreased cell viability, while palbociclib increased levels of nuclear p27, resulting in cell cycle arrest and higher cell viability. When the two drugs were combined, and gemcitabine was administered before palbociclib, the efficacy of the drugs was significantly increased and p27 expression was further downregulated. ☐ I also found that the effect of drug treatment resulted in increases in cleaved p27 that correlated with an enhancement of cell death. Reverse Transcriptase-qPCR the analysis revealed mRNA levels of CDKN1B (encodes p27) correlated with p27 protein expression. Further, by mass spectrometry analysis of p27 immunoprecipitated complexes, I identified novel p27 interacting proteins that are key components of signal transduction pathways for osteosarcoma. In summary, these data provide insight into the critical role p27 plays in osteosarcoma cell survival and identify novel p27 interactions that can be further studied as predictive biomarkers for drug response.