Abstract 4045: Hypoxia induces Plk4 expression and promotes immortalization of proliferating cells.
Authors: Rosario Carla , Swallow Carol J. 
Published: August 2014

Introduction: Polo-like kinase 4 (Plk4) is a serine/threonine kinase essential for centrosome duplication and cell cycle progression.High tumor expression of Plk4 is associated with poor prognosis in breast, pancreas and colorectal cancer patients. In addition, our laboratory has found evidence for a novel function of Plk4 in promoting cell migration. Endogenous Plk4 transiently localizes to cell protrusions during migration and spreading. Plk4 deficient cells show decreased spreading and migration potential, while Plk4overexpression enhances spreading and migration. While Plk4 is a haploinsufficient tumor suppressor in hepatocellular carcinoma, we hypothesize that Plk4 functions as an oncogene in promoting invasion in other cancer histologies.

Solid tumors develop with a microenvironment characterized by regions of chronically and transiently hypoxic cells. It has beensuggested that acute hypoxia drives tumor progression and facilitates metastasis. The objective of this project is to determine if hypoxic conditions affect Plk4 expression and Plk4-dependent cell phenotypes. Methods: Plk4+/+ and Plk4+/−murine embryonic fibroblast (MEFs) were cultured under hypoxic (3% oxygen)or normoxic (21% oxygen)conditions. Real time RT-PCR was used to quantify Plk4 expression. FACS analysis was used to determine ploidy. MEFs were passaged with either a 3T3 protocol (300,000 cells plated every 3d) or a 3T5 protocol (300,000 cells plated every 5d). At each passage, cells were counted, collected for RNA isolation and fixed for flow cytometry (n=2 embryos/genotype for each passaging protocol and condition). Results: Plk4+/+ and Plk4+/− MEFs show increased proliferation when cultured at 3% compared to 21% oxygen. Using a 3T5 protocol under normoxic conditions,Plk4+/+ cells undergo senescence by passage 8 whereas Plk4+/− cells become immortalized. These results are consistent with our previously published data (Rosario et al., PNAS 2010). Using a 3T3 protocol under normoxic conditions, Plk4+/+and Plk4+/−cells grow at a similar rate up to passage 5, after which they senesce. By contrast, during hypoxia, both Pk4+/+ and Plk4+/− MEFs immortalize with the 3T5 or 3T3 protocol. FACS analysis showed no apparent difference in ploidy between Plk4+/+ and Plk4+/−cells growing at 3% oxygen. Plk4 mRNA expression is induced under hypoxic conditions in both wildtype and heterozygous MEFs, compared to normoxic conditions. Conclusions: Culture at 3% oxygen alters the senescence program of MEFs, and induces Plk4 expression. Together, these changes may be sufficient to “rescue” the cytokinesis defect and development of aneuploidy otherwise observed in Plk4+/− MEFs immortalized with 3T5 passaging. The oscillating hypoxic microenvironment typical of solid tumors may facilitate tumor progression in part through induction of Plk4, with consequent augmentation of tumor cell proliferation, migration and metastatic potential.

Citation Format: Carla Rosario, Carol J. Swallow. Hypoxia induces Plk4 expression and promotes immortalization of proliferating cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4045. doi:10.1158/1538-7445.AM2013-4045