464453 Podocalyxin Knockdown Impairs Migration of Pancreatic Cancer Cells By Influencing Cytoskeletal Remodeling and Surface Adhesion
Podocalyxin Knockdown Impairs Migration of Pancreatic Cancer Cells by Influencing Cytoskeletal Remodeling and Surface Adhesion
Bin Sheng Wong, Daniel J Shea, Robert A Law, Konstantinos Konstantopoulos
Department of Chemical and Biomolecular Engineering, Johns Hopkins University
Introduction: Podocalyxin (PODXL) is a transmembrane sialomucin glycoprotein normally expressed in kidney podocytes1. While typically absent in normal pancreas, PODXL is often overexpressed in invasive pancreatic cancers and is associated with more aggressive tumor phenotypes2. Overexpression of PODXL has also been correlated to poor clinical prognosis of other cancers from breast3, colorectal4, renal5 and bladder6 origins. In this study, we aimed to investigate the effects of PODXL on the morphology, morphodynamics, migration and cytoskeletal dynamics of pancreatic cancer, and to elucidate the mechanism in which PODXL exerts its functions.
Materials & Methods: Stable clones of metastatic pancreatic adenocarcinoma SW1990 with reduced PODXL expression were generated using small hairpin RNA2. Knockdown (KD) efficiency was verified using western blot, flow cytometry and immunofluorescence staining. Random 2D and chemotactic-driven migration of PODXL scramble control (SC) and PODXL-KD SW1990 were assessed on collagen-I coated glass coverslips and microfluidic devices with microchannels of varying width respectively using time-lapse live microscopy. The cellsÕ migration, morphology and morphodynamic parameters were quantified using imageJ and a custom-written MATLAB program. Fluorescence recovery after photobleaching was conducted on cells transfected with LifeAct-GFP to quantify actin dynamics. Microtubule growth rates were examined via time-lapse confocal microscopy in cells transfected with end-binding protein 1-GFP. Cells were immunostained for phosphorylated paxillin and imaged with total internal reflection florescence microscopy to quantify focal adhesion size and density.
Figure 1: PODXL knockdown in SW1990 decreases their migration speed on 2D collagen-I coated glass surfaces (A) and reduces their velocity in microchannels across all channel widths (B). PODXL-KD slows down microtubule growth and dynamics (C). TIRF microscopy revealed diminished focal adhesion density in PODXL KD compared to SC (D). *²0.05, **²0.01, ***²0.001, ****²0.0001 by t-test.
Conclusion: PODXL knockdown markedly impairs migration of metastatic pancreatic cancer cells SW1990 on collagen-I coated 2D surfaces and in microchannels. These migratory deficiencies could be a result of interplay between reduced cellular morphodynamics, slower microtubule growth and higher focal adhesion density. Co-immunoprecipipation and mass spectroscopy assays failed to identify enrichment of ezrin, a widely reported binding partner of PODXL, in the immunoprecipitates of PODXL in pancreatic cancer cells. Experiments are underway to verify a novel cytoplasmic binding partner of PODXL that we have identified in pancreatic cancer cells and to uncover the exact molecular mechanisms by which PODXL exerts its pro-migratory effects.
References: 1Kerjaschki, D., et al. (1984) J Cell Biol 98(4): 1591-1596. 2Dallas, M. R., et al. (2012). Am J Physiol Cell Physiol 303(6): C616-624. 3Somasiri, A., et al. (2004). Cancer Res 64(15): 5068-5073. 4Larsson, A., et al. (2011). Br J Cancer 105(5): 666-672. 5Hsu, Y. H., et al. (2010). Am J Pathol 176(6): 3050-3061. 6Boman, K., et al. (2013). Br J Cancer 108(11): 2321-2328.