Abstract 5338: Pancreatic cancer tumor initiating cells are marked by the presence of cell surface associated acetylated alpha tubulin

Abstract

Abstract The function of tubulin acetylation in the context of cancer initiation and progression remains unknown. In studying the frequency of primary cilia in early pancreatic cancer, we discovered an increase in acetylated alpha-tubulin labeling (AAT) labeling in murine PanIN, even in the absence of primary cilia. We therefore hypothesized that human pancreatic ductal adenocarcinoma (PDAC) cells may upregulate expression of AAT as they progress from non-invasive to invasive and metastatic disease. Performing confocal microscopy on a human PanIN (n=23) and metastatic PDAC (n=10) tissue arrays, we have determined that in 15% of PanIN lesions, 13-30% of the cells have detectable levels of AAT, with a primarily cytoplasmic/cell membrane pattern of labeling. In contrast, over 80% of metastatic PDAC cells are AAT-positive. Surprisingly, high-resolution 3D confocal imaging of both fixed and unfixed human PDAC cell lines demonstrated immunoreactive AAT to be present on the cell surface of unfixed cells, as previously reported in human leukemia cells (Quillen, JCB 1985). To evaluate the functional significance of this finding, we developed a FACS-based strategy to isolate viable PDAC cells based on levels of cell surface AAT. Applying this strategy to four different human pancreatic cancer cell lines and three early passage human xenografts, we have determined that 5-10% of human pancreatic cancer cells have detectable levels of cell-surface AAT. PDAC cells positive for cell surface AAT are markedly enriched in cells also positive for either CD133 or CD24/CD44, representing putative pancreatic cancer stem cell populations. We have functionally analyzed the tumor-initiating capacity of cell surface AAT+ cells using in vitro tumor sphere experiments. In this assay, cell surface AAT+ cells show a statistically significant 100-fold increase in tumor sphere-forming capacity relative to cell surface AAT-negative cells. Tumor sphere-forming capacity was significantly reduced by neutralizing anti-AAT antibodies, as well as by an siRNA targeting α-tubulin K40 acetyltransferase (αTAT). We further evaluated the functional significance of cell surface AAT by analyzing the in vivo tumor initiating capacities of cell surface AAT-positive and AAT-negative cells from a human PDAC xenograft. Cell surface AAT-positive cells exhibited significantly increased tumor initiating frequency relative to the AAT-negative population (**p<0.0000539). AAT-positive cells had a tumor-initiating frequency similar to an Aldefluor-bright population, suggesting that the presence of cell surface acetylated alpha tubulin may mark a pancreatic cancer tumor initiating population. These data provide important new information regarding cellular heterogeneity in human pancreatic cancer, and suggest that novel strategies targeting cell surface tubulin may have therapeutic utility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5338. doi:1538-7445.AM2012-5338