(776a) Podocalyxin Knockdown Impairs Migration of Pancreatic Cancer Cells By Influencing Cytoskeletal Remodeling and Surface Adhesion

Wong, B. S., Johns Hopkins University
Konstantopoulos, K., Johns Hopkins University
Shea, D., Johns Hopkins University
Law, R., Johns Hopkins University

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

IntroductionPodocalyxin (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.

20160425 BMES Abstract Figures 2.tiffMaterials & 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.

" src="https://www.aiche.org/sites/default/files/aiche-proceedings/conferences/153011/papers/464453/Paper_464453_abstract_90462_0.png" height="101" hspace="9" class="documentimage">Results
& Discussions
: PODXL was successfully
knocked down (>90%) in SW1990 cells, resulting in decreased PODXL surface
coverage and membranous localization. Migration of PODXL-KD SW1990 is
significantly reduced compared to the SC on both 2D (Fig. 1A) and in microchannels
of all widths (Fig. 1B). PODXL-KD
cells are generally smaller, less elongated, more circular, less protrusive and
exhibit much slower morphodynamics compared to the SC.
While displaying similar actin dynamics, PODXL knockdown significantly slows
down the rate of microtubule growth (Fig.
), thereby contributing to diminished morphodynamics
and reduced migration. Furthermore, PODXL-KD cells possess significantly higher
focal adhesion density compared to the SC despite having comparable focal
adhesion size (Fig. 1D). Elevated
focal adhesion density may lead to increased traction forces in the PODXL-KD
cells which enhances their adhesion to the surfaces, thereby impeding efficient

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.

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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.