Invadopodia are actin-rich structures at the base of many neoplastic cells that sequester matrix metalloproteases that act to degrade the surrounding stroma to facilitate the invasive process. Conventional invadopodia are dependent upon Src kinase and the large GTPase dynamin 2 (Dyn 2). Whether invadopodia are the only mechanism by which cells degrade matrix is unclear. We have observed that cells of mesenchymal origin degrade matrix in an unique way different from tumor cells. The HYPOTHESIS of this study is that fibroblasts, and other cells of mesenchymal origin, degrade matrix by a mechanism distinct from that of epithelial-based tumor cells. The CONCLUSION is that stromal cells degrade matrix by a novel mechanism distinct from traditional invadopodia

Cao, Hong

Chen, Jing

Eppinga, Robbin

Krueger, Eugene W.

McNiven, Mark A.

English

Dordt College

Digital Collections @ Dordt Faculty Work Comprehensive List 12-18-2012 Large Scale Matrix Degradation by Stromal Cells Independent of Invadopodia Hong Cao Mayo Clinic Robbin Eppinga Dordt College, robbin.eppinga@dordt.edu Eugene W. Krueger Mayo Clinic Jing Chen Mayo Clinic Mark A. McNiven Mayo Clinic Follow this and additional works at: https://digitalcollections.dordt.edu/faculty_work  Part of the Cancer Biology Commons Recommended Citation Cao, H., Eppinga, R., Krueger, E. W., Chen, J., & McNiven, M. A. (2012). Large Scale Matrix Degradation by Stromal Cells Independent of Invadopodia. Retrieved from https://digitalcollections.dordt.edu/faculty_work/187 This Conference Presentation is brought to you for free and open access by Digital Collections @ Dordt. It has been accepted for inclusion in Faculty Work Comprehensive List by an authorized administrator of Digital Collections @ Dordt. For more information, please contact ingrid.mulder@dordt.edu. Large Scale Matrix Degradation by Stromal Cells Independent of Invadopodia Abstract Invadopodia are actin-rich structures at the base of many neoplastic cells that sequester matrix metalloproteases that act to degrade the surrounding stroma to facilitate the invasive process. Conventional invadopodia are dependent upon Src kinase and the large GTPase dynamin 2 (Dyn 2). Whether invadopodia are the only mechanism by which cells degrade matrix is unclear. We have observed that cells of mesenchymal origin degrade matrix in an unique way different from tumor cells. The HYPOTHESIS of this study is that fibroblasts, and other cells of mesenchymal origin, degrade matrix by a mechanism distinct from that of epithelial-based tumor cells. The CONCLUSION is that stromal cells degrade matrix by a novel mechanism distinct from traditional invadopodia. Keywords invadopodia, tumor cells, stromal cells, cancer Disciplines Cancer Biology | Cell and Developmental Biology Comments Poster presented at the American Society for Cell Biology Annual Meeting in San Francisco, California, December 15-19, 2012. Abstract #2075 This conference presentation is available at Digital Collections @ Dordt: https://digitalcollections.dordt.edu/faculty_work/187 Large Scale Matrix Degradation by Stromal Cells Independent of Invadopodia  Hong Cao, Robbin Eppinga, Eugene W. Krueger, Jing Chen, and Mark A. McNiven  Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota USA  Biology Department, Dordt College, Sioux Center, Iowa USA       a c Figure 6. Matrix Degradation by Epithelial Cells and Fibroblasts Exhibits Distinct Requirements for Src Kinase   Figure 5. Knockdown of Dyn 2 Induces a Modest  Retention of MT1-MMP on the Cell Surface     Invadopodia are actin-rich structures at the base of many neoplastic cells that  sequester matrix metalloproteases that act to degrade the surrounding stroma  to facilitate the invasive process.  Conventional invadopodia are dependent upon  Src kinase and the large GTPase dynamin 2 (Dyn 2).  Whether invadopodia are  the only mechanism by which cells degrade matrix is unclear.  We have observed  that cells of mesenchymal origin degrade matrix in an unique way different from  tumor cells.        The HYPOTHESIS of this study is that fibroblasts, and other cells of  mesenchymal origin, degrade matrix by a mechanism distinct from that  of epithelial-based tumor cells.     The GOALS of this study were to define the mechanisms supporting  this novel form of matrix remodeling by:   - Defining the requirements of Dyn 2 and Src kinase in matrix degradation by    fibroblasts and tumors of epithelial origin.   - Testing the role of Dyn 2 depletion on MT1-MMP accumulation by cells both     internally and at the plasma membrane.    Background and Goals Figure 1. Tumor Cells and Stromal Cells Exhibit Distinct  Patterns of Matrix Degradation  A variety of different tumor and mesenchymal cell types plated on fluorescent gelatin and  cultured for 8h prior to fixation. (a,b) The pancreatic tumor cells BxPC3 and DanG show  largely invadopodial-like (white arrowheads) and focal adhesion-like (arrows) degradation  patterns. (c) The fibrosarcoma cell line HT1080 exhibits focal adhesion-like degradations and  a unique pattern of centrally positioned degradation structure in a labyrinth or reticular-like  display. (d-f) Mesenchymal cells exhibit a unique pattern of matrix degradation, whether they  be rat fibroblasts, human fibroblasts, or pancreatic stellate cells. (g) Graph depicting the  percentage of each cell type that degrades matrix after 8 hours. Figure 2. Reduction of Dynamin 2 (Dyn 2 ) Induces a  Differential Response in Matrix Degradation Between  Fibroblasts and Epithelial Cells   Figure 3. Tamoxifen-Induced Knockout of Dyn 2 in MEFs Induces a Substantial Increase of Matrix Degradation Compared to Controls  To examine another fibroblast cell line from a Dyn 2 genetic knockout model (kindly  provided by Ferguson and De Camilli, Yale), matrix degradation was analyzed in MEFs  from these knockout mice in the presence and absence of tamoxifen. (a,b) Immunostaining  of control or tamoxifen-treated cells (4-5 days) stained for Dyn 2, or revealing matrix  degradation on a fluorescent gelatin substrate. Control cells show substantial amounts of  endogenous cytoplasmic Dyn 2 but exhibit almost no matrix degradation. In contrast, cells  in which Dyn 2 has been reduced exhibit a 6-7-fold increase in matrix degradation with a  characteristic reticular network pattern. (d) Western blot analysis of cells +/- tamoxifen  treatment blotted for Dyn 2. (e-g) Re-expression of either Dyn 1 or Dyn 2 in tamoxifen- induced Dyn 2 knockout MEFs results in a reversal of the degradation phenotype (reduced  degradation). Fluorescence images showing re-expression of either of the neuronal-specific  Dyn 1 or ubiquitously expressed Dyn 2 with corresponding degradation patterns on  fluorescent gelatin. Note that the surrounding cells in which dynamin is not expressed  degrade large amounts of matrix in comparison to the rescued cells (*) that degrade no  matrix at all. (g) Quantitation of the effect of dynamin knockout on matrix degradation  by MEFs. Note the substantial increase in the tamoxifen-treated Dyn KO cells, which is  markedly reduced by expression of wild type Dyn 2 and Dyn 1-GFP. In contrast, the  Dyn 2K44A mutant-expressing cells continue to degrade large amounts of matrix.   Western blot analysis of six different mesenchymal and epithelial cell lines in which  endogenous Dyn 2 levels have been experimentally reduced, followed by SDS-PAGE  and Western blot analysis with antibodies to MT1-MMP. (b) Quantitation of three  distinct experiments shows a modest accumulation of MT1-MMP protein upon Dyn 2  knockdown. While levels were increased by nearly 50% in the RF, DKO MEFS, and  BxPC3 cells, there was only a modest incease observed in the PSC cells and an actual  loss of protease in the DanG cells.  Figure 4. A Reduction of Dyn 2 Causes Accumulation  of MT1-MMP  Conclusions Stromal cells degrade matrix by a novel mechanism distinct from traditional  invadopodia.  Summary In this study we have found that:   •  Fibroblasts and cells of mesenchymal origin degrade matrix but appear     to do so by a mechanism distinct from epithelial-based tumor cells.   •  Matrix degradation in fibroblasts appears to be mediated by focal     adhesion sites or at a reticular structure at the cell base.  Epithelial     tumor cells degrade by both focal adhesions and traditional invadopodia.   •  Matrix degradation by invadopodia in epithelial tumor cells is markedly     reduced by Dyn2 knockdown or inhibition of Src kinase while degradation     by fibroblasts is increased substantially by Dyn 2 depletion and is Src     independent.    •  Reduction in Dyn2 levels results in a 2 fold increase of MT1-MMP with     a modest proportion of this protease remaining at the cell surface.  (a,c) Western blot analysis of cell homogenates of either rat fibroblasts treated with  siRNA to knockdown dynamin, or MEFs treated with tamoxifen to induce the Dyn 2 knockout.  Following reduction of Dyn 2 levels, cells were incubated in biotin to label all cell surface  proteins, then homogenized followed by a pull-down with streptavidin beads, then SDS-PAGE  and Western blot (see cartoon for experimental sequence). (b,d) Graphs represent   quantitation of three different experiments showing both total, and surface populations of  MT1-MMP from cells both before and after Dyn2 knockdown. As shown before in Fig. 4, total  MT1 levels were increased by 2 fold, although changes in the levels of MT1 residing at the  cell surface remain only modestly increased (20-40%) following Dyn2 knockdown.  This minor  increase suggests that the massive increase in matrix degradation by the mesenchymal cells  following Dyn2 reduction may not be dependent upon an accumulation of active protease at  the cell surface.  Assessing the effects of Dyn2 knockdown on matrix degradation in six different fibroblast  and epithelial cell types. Cells were treated with siRNA to reduce Dyn2 levels over 72 h, then  trypsinized, resuspended, and plated on fluorescent gelatin-coated coverslips for six hours  in the presence of the protease inhibitor BB94. Cells were then rinsed and allowed to degrade  for 8 hrs prior to fixation. (a-e) A variety of mesenchymal fibroblastic cells that normally  exhibit modest amounts of degradation show increased matrix remodeling by 4-5-fold upon  reduction of Dyn 2. While some focal adhesion-like degradation (white arrows) is observed,  most of it is a reticular-like degradative pattern at the center of the cell. In contrast,  pancreatic epithelial tumor cells that spontaneously degrade via invadopodia are markedly  inhibited in their degradative capacity by Dyn2 knockdown (g-i). (j) Western blot analysis of  cell homogenates showing near complete elimination of Dyn2 expression in the six cell types  examined.     siRNA Invadopodia function is dependent upon Src kinase. To determine if the distinct matrix  degradation processes observed in the different cell types shared a requirement for Src  kinase, we treated cells with the Src inhibitor PP2. Cells were plated on gelatin in the presence  of the MMP inhibitor BB94 for 5 hours during plating to prevent degradation during attachment.   Subsequently cells were washed free of BB94 and incubated in 25mM PP2 for 13 hours. (a, e)  The pancreatic cancer lines DanG and BxPC3 both show robust degradation under control  conditions, but are almost completely inhibited when treated with 25mM PP2 (b, f). By contrast,  the fibrosarcoma cell line HT108 (c, d) and the Dyn2 knockout fibroblast cell line DKO (g, h)  both continue to degrade large amounts of matrix even under these high concentrations of  inhibitor.  (i) A graph depicting the percentage of cells that degrade matrix upon Src inhibition.  Note the graphic contrast in drug sensitivity between the epithelial tumor cells and the  fibroblasts/fibroscarcoma cell lines. 

Large Scale Matrix Degradation by Stromal Cells Independent of Invadopodia

https://digitalcollections.dordt.edu/cgi/viewcontent.cgi?article=1186&context=faculty_work

Large Scale Matrix Degradation by Stromal Cells Independent of Invadopodia

Abstract

Similar works

Full text

Available Versions

Dordt College