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

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 (AcTub) 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 AcTub as they progress from noninvasive 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 AcTub, with a primarily cytoplasmic/cell membrane pattern of labeling. In contrast, over 80% of metastatic PDAC cells are AcTub-positive. Surprisingly, high-resolution 3-D confocal imaging of both fixed and unfixed human PDAC cell lines demonstrated immunoreactive AcTub to be present on the cell surface of unfixed cells, as previously reported in human leukemia cells (Quillen, JCB 1985). Furthermore, TEM imaging of cancer cells revealed the expression of AcTub on the cell membrane. 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 AcTub. 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 AcTub. PDAC cells positive for cell surface AcTub 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 AcTubHI cells using in vitro tumor sphere experiments. In this assay, cell surface AcTubHI cells show a statistically significant 100-fold increase in tumor sphere-forming capacity relative to cell surface AcTublow cells. Tumor sphere-forming capacity was significantly reduced by neutralizing anti-AcTub antibodies, as well as by an siRNA targeting α-tubulin K40 acetyltransferase (αTAT). We further evaluated the functional significance of cell surface AcTub by analyzing the in vivo tumor initiating capacities of cell surface AcTubHI and AcTublow cells from a human PDAC xenograft. Cell surface AcTubHI cells exhibited significantly increased tumor initiating frequency relative to the AcTublow population (**p<0.0000539). AcTubHI 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. This work has been supported by the PanCAN-AACR Pathway to Leadership Award to JMB and NIH CA158898 to SDL. Citation Format: Jennifer M. Bailey, Janivette Alsina, Florencia McAllister, Zeshaan Rasheed, Anirban Maitra, William Matsui, Steven Leach. Pancreatic cancer tumor-initiating cells are marked by the presence of cell surface-associated acetylated alpha tubulin. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B55.