Abstract 5144: Ubiquitin carboxy-terminal esterase L1 (UCHL1) in stem-like cancer cells of embryonal neural tumors

Abstract

Abstract Emerging evidence suggest that most cancers have “stem cells” and that these stem-like cells are the drivers of tumor growth and metastases. Therefore targeting of these cancer stem cells will lead to advances in treatment of the disease. Accordingly, a rational option is to interfere with the function and or expression of genes or proteins that are essential for the self-renewal and expansion of the cancer stem cell fraction. We performed proteomic analysis to compare and identify proteins, the level of which is differentially altered between the neuropsheres (enriched cancer stem cell fraction) and the adherent cells, of human medulloblastoma cell line Daoy and human neuroblastoma cell line SK-N-BE(2). Several proteins important in cell cycle control, signal transduction, and cell differentiation were found to be over expressed in the cancer stem cell enriched neuropsheres. Intriguingly, we identified a deubiquitinating enzyme UCHL1 (ubiquitin carboxyl-terminal esterase L1) to be over-expressed in the neurospheres. UCHL1 is a component of the ubiquitin-proteasome system, the major machinery responsible for intracellular protein degradation and regulation of many key biological processes. The present study was undertaken to determine the role of UCHL1 in cancer stem cell functions. The differential level of UCHL1 was validated using western blot analyses in SK-N-BE(2), SH-SY5Y and Daoy cell lines. The levels of UCHL1 in the neurosphere culture of SKNBE(2), Daoy and SH-SY5Y were ∼1.6, 1.7, and 2.0 fold respectively, higher, than the adherent culture, thus confirming our proteomic results. The mRNA level of UCHL1 was determined using quantitative RT-PCR. There was no significant difference in expression of mRNA levels of UCHL1 between the adherent cultures and neurospheres cultures therefore suggesting the possibility of a post-transcriptional regulation of UCHL1. Blockade of the hydrolase activity of UCHL1 was performed using small molecule inhibitor LND-5744. IC50 was determined in Daoy and SK-N-BE(2) neurospheres and monolayer adherent cells. Interestingly, both Daoy and SK-N-BE(2) neurospheres were more sensitive to LND-5744 than the adherent cells (Daoy neurosphere IC50 ∼100µM vs Daoy adherent cells ∼125µM and SK-N-BE(2) neurosphere IC50 ∼10-25µM, vs SK-N-BE(2) adherent cells ∼100µM). Thus, its loss seems to have a detrimental effect in the ability of cancer stem cell self-renewal, readout as impairment in neurosphere formation in the presence of LND-5744. Additional functional validation using small interfering RNA silencing of UCHL1 to confirm that its elevated expression may contribute to cancer stem cell properties are ongoing. The findings from this study suggested that UCHL1 might serve as prospective molecular target for neural embryonic tumors. 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 5144.