Abstract #4535: Effects of the chemical genetic inhibition of LMP2 in prostate cancer cells

The purpose of these studies is to elucidate the functional effects of inhibiting the immunoproteasome subunit LMP2 in prostate cancer cell lines using a novel chemical genetic tool, UK-101, developed in our laboratory, and to then compare these effects to those of a widely acting proteasome inhibitor, epoxomicin. Prostate cancer cell lines used in this study either express a relatively high level of the LMP2 subunit (PC3) or a relatively low level (LNCaP). The expression of LMP2 was increased in these cell lines using a natural inducer of the immunoproteasome (interferon-gamma). Dose- and time-dependent expression levels of LMP2 were determined via western blotting. These characterized cells were then treated with UK-101 or epoxomicin and the effects of each of these proteasome inhibitors on cell proliferation were measured using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS assay). Additionally, PC3 cells were treated with UK-101, epoxomicin, or vehicle and subjected to 2D gel electrophoresis with silver staining to observe the differential expression of proteins due to blockage of the subunit LMP2 or X, respectively. Proteins whose differential expression was significant between vehicle and compound on the silver gel were then matched to spots on a Coomassie stained gel. These matched spots were then subjected to mass spectral analysis for protein identification. Expression of LMP2 can be upregulated by interferon-gamma in PC3 cells in a dose-dependent manner and the upregulation lasts for at least 96 hours. Expression of LMP2 was also upregulated in LNCaP cells in a dose-dependent manner. The preliminary evidence indicates that increasing the expression of LMP2 in PC3 cells has no significant effect on the potency of UK-101 or epoxomicin, while the effects of UK-101 and epoxomicin on LNCaP cells treated with interferon-gamma will also be presented. Inhibition of LMP2 causes the accumulation of proteins, some of which are unique when compared to those found upon inhibition of X. The identity of these proteins will provide promising leads for future mode of action studies of UK-101. LMP2 expression levels correlate with the sensitivity of prostate cancer cells to UK-101. By manipulating the LMP2 expression level in these cells using a natural inducer, we have modulated the expression of the immunoproteasome and examined the relationship between LMP2 and UK-101-induced cytotoxicity without the confounding effects of comparing different cell lines. Our chemical genetics approach using UK-101 and epoxomicin also has the potential to clarify the existing controversy as to the independent and overlapping functions of the constitutive and immunoproteasomes.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4535.