(725f) Loss of Giant Obscurins Enhances Migration and Cell Dynamics in Pancreatic Ductal Epithelial Cells

Authors: 
Shea, D., Johns Hopkins University
Konstantopoulos, K., Johns Hopkins University
Kontrogianni-Konstantopoulos, A., University of Maryland School of Medicine

Loss of Giant Obscurins
Enhances Migration and Cell Dynamics in Pancreatic Ductal Epithelial Cells

Daniel J Shea1,
Konstantinos Konstantopoulos1, and Aikaterini Kontrogianni-Konstantopoulos2

1Department of Chemical and Biomolecular
Engineering, Johns Hopkins
University, 2Department of
Biochemistry and Molecular Biology, University of Maryland School of Medicine.

Introduction: Obscurins are giant cytoskeletal proteins with structural and regulatory
roles that are encoded by the gene OBSCN.
Depletion of giant obscurins from breast epithelial cells has been correlated with
increased apoptotic resistance1, epithelial to mesenchymal
transition, tumorigenicity, and metastasis2.
Additionally, mutations in the OBSCN
gene have been associated with pancreatic ductal adenocarcinoma3, 4.
Since
pancreatic cancer has the lowest (5-year) survival rate of any major
malignancy, and metastasis is the single most important contributor to this
prognosis, we looked to assess the role
of obscurins in pancreatic cancer metastasis. We investigated how depletion of
obscurins from human pancreatic ductal epithelial (HPDE) cells affects cell migration and cytoskeletal
dynamics using a series of microchannel
migration assays and real-time measurements of actin, microtubule, and focal
adhesion (FA) turn-over rates.

Materials and Methods: Immunoblotting was utilized to assess the obscurin levels in six
pancreatic cell lines. A pancreatic tissue microarray was stained for obscurin
alongside with obscurin-expressing HPDE cells, and imaged via confocal microscopy
to determine obscurins localization and expression. HPDE cells were transfected
with shRNA constructs targeting the OBSCN gene or control (scramble) shRNA, and stable cell lines were produced. Cross-shaped
PDMS microchannels with a base channel (10µm)
bifurcating to three-branch channels with varying widths of 3, 6 and 10µm (the
10µm device shown in Fig 1A) were fabricated using photolithography. Cells were
tracked as they entered the channel bases until they made a decision at the
bifurcation point and continued to the branch regions. Additionally, actin
dynamics at cell-cell junctions were measured using fluorescence recovery after
photobleaching (FRAP) for LifeAct-RPF
transfected HPDE cells. Microtubule growth was examined using time-lapse
confocal microscopy to image EB1-GPF transfected HPDE cells. FA density was
measured via TIRF microscopy. RhoGTPase pull-down was
performed to assess the levels of active rhoA, given
that the RhoGEF motif of obscurins specifically binds
and activates RhoA. All experiments were
performed using obscurin-KD and
control HPDE cells.

Results and
Discussion:
Pancreatic tissue microarray analysis shows high
obscurin expression in normal pancreatic tissue and reduced expression in
pancreatic adenocarcinoma. Using immunoblotting,
giant obscurins were identified in epithelial but not mesenchymal
pancreatic cell lines, and HPDE cells were found to express the highest
levels of obscurins. Confocal
images of HPDE cells show that obscurins are localized in the cytosol, and are
absent from cell-cell junctions when grown in the absence of FBS.  In the cross-shaped microchannels,
obscurin-KD HPDE cells migrated with a higher velocity in the channel base,
made a decision in the bifurcation region more quickly (Fig 1A), were more
protrusive (lower solidity), and less circular than control cells, indicating that they were more
dynamic. Coinciding with a pronounced decrease in active rhoA
levels (Fig 1C), obscurin-KD HPDE cells were found to have faster (more
dynamic) microtubule growth (Fig 1B) and decreased FA density. Furthermore,
actin dynamics at cell-cell
junctions were only increased for obscurin-KD cells in the presence of 10% FBS,
where obscurin localization shifted from cytosol to the cell-cell junctions.


Figure 1. (A) Obscurin-KD HPDE cells exhibit faster decision-making time in cross-shaped device, (B) increased microtubule dynamics and (C) have less active rhoA when compared to control cells. (*P<.05, ****P<.0001)

 

BMES2.tif

Conclusions: Given that one of the major hallmarks of pancreatic
cancer is its extensive local tumor invasion, and its dissemination to distant
organs,
identifying biomarkers that play a role in pancreatic cancer metastasis is
vital to the treatment and prevention of the disease. We found that the loss of
giant obscurins from epithelial pancreatic cells results in a more migratory
and dynamic cell phenotype. Furthermore,
in
addition to comprehensively exploring cell migration and cytoskeletal
properties, we have also begun exploring the effects of obscurinsÕ depletion in vivo using orthotopic
pancreatic implantation of cells in mice.

References: (1) Perry,
N.A. et al., FASEB
J,
 2012, 26,7.  (2)
Shiver M. et al., Oncogene, 2014, 34(32), 4248-4259 (3) Hocker, J. R., et al., Cancer Lett, 2015, 359, 2. (4) Murphy, S. J., et al., Gastroenterology 2013, 145, 5.