Abstract 2624: Targeted inhibition of the immunoproteasome subunit LMP2 using UK-101 induces apoptotic cell death in colorectal cancer

Abstract

Abstract Background: The immunoproteasome is an alternative form of the constitutive proteasome and it has been increasingly shown to play an important role in a number of diseases including cancer and inflammatory diseases. We have previously developed ‘UK-101’, a small molecule that selectively inhibits the immunoproteasome catalytic subunit LMP2. In our present study, we investigated the frequency of LMP2 expression in primary colon cancers and the impact of targeted inhibition of LMP2 on colon cancer cell survival and apoptosis using UK-101. Methods: The levels of LMP2 expression were assessed in colon cancers and adjacent nonmalignant colonic tissues from the matching donors via immunoblotting and immunohistochemical (IHC) analyses. The frequency of LMP2 expression was further determined by performing IHC staining of LMP2 on a tumor tissue microarray (TMA) containing 153 colorectal adenocarcinomas. Using colon cancer cell lines expressing LMP2 (SW480 and DLD-1), we have investigated the effect of UK-101 on tumor cell survival and apoptosis using the MTS and TUNEL assays. To probe the molecular mechanisms of UK-101-induced cell death, we have compared the effects of UK-101 on a number of apoptosis-related proteins and mediators in apoptotic pathways with those of the broadly acting proteasome inhibitors (bortezomib and epoxomicin). Results: Our immunoblotting and IHC analyses indicated that the LMP2 levels were substantially upregulated in colon cancers compared to the adjacent nonmalignant colonic tissues from the matching donors. The IHC staining on TMA also demonstrated positive immunostaining for LMP2 in approximately 70% of colon cancer specimens evaluated (n=107 out of 153). A number of colon cancer cell lines were found to express LMP2 at differing levels. Using DLD-1 and SW480 cells expressing LMP2 at moderate to high levels, we observed that UK-101 covalently modifies LMP2, but not other catalytic subunits. The UK-101 treatment also induced cell death, measured by the MTS assay and TUNEL staining. Consistent with apoptotic cell death and proteasomal inhibition, UK-101 treated cells showed the cleavage of PARP and caspase-3 and accumulation of polyubiquitinated proteins. Interestingly, the UK-101 treatment did not show any inhibitory effect on the NF-kB pathway, which was observed with bortezomib and epoxomicin. Conclusion: Our results show that the targeted inhibition of LMP2 using UK-101 induces apoptotic cell death in colon cancer. UK-101 does not show any inhibitory effect on the NF-kB pathway in colon cancer cells, suggesting that UK-101 induces colon cancer cell apoptosis via mechanisms different from bortezomib and epoxomicin. Taken together, these results suggest that LMP2 may be a potential therapeutic target in treating colon cancer. Further studies are ongoing to investigate the signaling pathways related to UK-101 induced cell death in colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2624.