Mutant p53 regulates ovarian cancer transformed phenotypes through autocrine matrix deposition
Marcin P. Iwanicki, … , Ronny Drapkin, Joan S. Brugge
Marcin P. Iwanicki, … , Ronny Drapkin, Joan S. Brugge
Published July 7, 2016
Citation Information: JCI Insight. 2016;1(10):e86829. https://doi.org/10.1172/jci.insight.86829.
View: Text | PDF
Research Article Cell biology Oncology

Mutant p53 regulates ovarian cancer transformed phenotypes through autocrine matrix deposition

Abstract

High-grade serous ovarian carcinoma (HGS-OvCa) harbors p53 mutations and can originate from the epithelial cell compartment of the fallopian tube fimbriae. From this site, neoplastic cells detach, survive in the peritoneal cavity, and form cellular clusters that intercalate into the mesothelium to form ovarian and peritoneal masses. To examine the contribution of mutant p53 to phenotypic alterations associated with HGS-OvCA, we developed live-cell microscopy assays that recapitulate these early events in cultured fallopian tube nonciliated epithelial (FNE) cells. Expression of stabilizing mutant variants of p53, but not depletion of endogenous wild-type p53, in FNE cells promoted survival and cell-cell aggregation under conditions of cell detachment, leading to the formation of cell clusters with mesothelium-intercalation capacity. Mutant p53R175H-induced phenotypes were dependent on fibronectin production, α5β1 fibronectin receptor engagement, and TWIST1 expression. These results indicate that FNE cells expressing stabilizing p53 mutants acquire anchorage independence and subsequent mesothelial intercalation capacity through a mechanism involving mesenchymal transition and matrix production. These findings provide important new insights into activities of mutant p53 in the cells of origin of HGS-OvCa.

Authors

Marcin P. Iwanicki, Hsing-Yu Chen, Claudia Iavarone, Ioannis K. Zervantonakis, Taru Muranen, Marián Novak, Tan A. Ince, Ronny Drapkin, Joan S. Brugge

×

Figure 1

Mutant p53 promotes survival of detached fallopian tube nonciliated epithelial (FNE) cells.

Options: View larger image (or click on image) Download as PowerPoint
Mutant p53 promotes survival of detached fallopian tube nonciliated epit...
(A) Montage of representative (n = 8–10/group) phase-contrast video clips from time-lapse recording of FNE cells transduced with empty vector or mutant p53 (m-p53) variants (R175H, R249S, or R273H) and maintained in suspension for the indicated time. Arrows point to dying cells. Clips are representative of 8–10 movies per condition with 100–150 cells per movie acquired during one recording session (Supplemental Video 1). (B) Schematic depiction of the ethidium bromide (EtBr) incorporation assay used to quantify cell death (red) within multicellular clusters (green) grown in suspension. (C and D) Representative (n = 3–4 experiments) phase-contrast and pseudocolored fluorescence images documenting the level of EtBr incorporation into GFP-labeled FNE vector control (pWZL in C, pLenti 6 in D) or FNE-m-p53 (R175H, R249S, and R273H) cellular clusters maintained in suspension for 96 hours. (E) Quantification of the EtBr incorporation distribution in cell clusters from 4 (R175H) and 3 (R249S and R273H) independent experiments. n = 50–60 cellular clusters in control and m-p53R175H groups, and n = 61, 42, and 30 cellular clusters in control, m-p53R249S, and m-p53R273H groups, respectively. Each cluster contained 100–150 cells. Scale bar: 150 μm. All data shown as the median (horizontal bar), interquartile range (box), and minimum/maximum values (whiskers). Statistical analysis was performed using either 2-tailed Student’s t test or 1-way ANOVA and post-hoc Tukey-Kramer test. *P < 0.05 for each p53 mutant cell line relative to the vector control.