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

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 adenocarcinoma^{3, 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)

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.