Analysis of Antizyme Inhibitor Function in Prostate Tumor Growth and Centrosome Amplification

Background: Antizyme inhibitor (AZI) is an endogenous protein which promotes cell proliferation and increased polyamine levels by interfering with antizyme (AZ)-mediated degradation of ornithine decarboxylase (ODC). Our lab has also shown that both AZ and AZI localize to centrosomes in mammalian cells and can modulate centriole duplication. The purpose of this study was to explore the mechanism by which AZI contributes to tumorigenesis. Specifically, to determine whether AZI has an important functional role during tumor growth in vivo, and to explore how changes in levels of AZ and AZI impact centrosome duplication.;Methods: To examine the importance of AZI in tumor growth, I made prostate (PC3M-LN4, AT6.1) and kidney (Hek293) cancer cell lines stably expressing either shRNA to knockdown AZI or control shRNA. shAZI cell lines had significantly decreased levels of both AZI and ODC protein, and decreased cell proliferation in vitro. shAZI and shGFP control cells were injected subcutaneously into nude mice (1x106 cells/mouse) and tumor size was measured. To explore how AZ and AZI impact centrosome duplication, first I analyzed the effect of AZI overexpression on centriole formation. I also examined the ability of AZ to interact with proteins known to induce daughter centriole amplification using immunoprecipitation studies and direct binding assays. Lastly, I generated Tet-inducible reagents to overexpress AZI, AZ, ODC, or selected proteins involved in regulating centrosome duplication.;Results: AZI knockdown significantly decreased proliferation of both PC3M-LN4 and AT6.1 cell lines in vitro. Intriguingly, shAZI cells displayed an even greater reduction of tumor growth in vivo compared to shGFP cells. Our data also suggests that AZ may interact with NDR1 kinase and Plk1 kinase, two proteins known to be important for centrosome duplication.;Conclusions: Our results suggest that AZ1 levels contribute to efficient growth of prostate cancer tumors, and therefore in vivo suppression of AZI may be beneficial for prostate cancer treatment. Further studies are needed to fully elucidate how changes in AZ levels impact both NDR1 and Plk1. Together, these studies suggest that AZI may contribute to tumorigenesis not only by increasing cell proliferation, but also by affecting centrosome duplication.