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.
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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

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Figure 6

Mutant p53 (m-p53) promotes fibronectin secretion and mesenchymal transcription in fallopian tube nonciliated epithelial (FNE) cells.

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Mutant p53 (m-p53) promotes fibronectin secretion and mesenchymal transc...
(A) Upper panel: Western blots of fibronectin, p53, and actin expression in FNE-m-p53R175H cells cultured in suspension for 24 hours. Fibronectin and actin blots were repeated 3 times. Lower panel: Western blot of secreted fibronectin from trichloroacetic acid–precipitated protein preparation from serum-free 24-hour suspension cultures of various FNE cell lines. Actin blotting was performed on whole-cell lysate. This experiment was performed twice. (B) Confocal microscopy images of fibronectin immunofluorescence staining (red) and GFP (green) in the various FNE cell lines cultured in suspension. Scale bar: 100 μm. Inset shown at higher magnification. Scale bar: 50 μm. (C) Confocal microscopy images of integrin β1 and fibronectin immunofluorescence staining in FNE-m-p53R175H cell clusters cultured in suspension for 72 hours. Scale bar 50: μm. Arrows point to outer-surface localization of integrin β1 and fibronectin. (D) Upper panel: Western blots of fibronectin, laminin C1, integrin α5, E-cadherin, and actin expression among various DF samples. This experiment was repeated twice. Lower panel: Confocal images of fibronectin and activated integrin β1 immunofluorescence staining in DF cell clusters cultured in suspension for 72 hours. Arrows point to fibronectin- and active β1 integrin–rich plaques detected on the outer cell surface. Scale bar: 150 μm. (E) Twist family BHLH transcription factor 1 (TWIST1), Snail family zinc finger 1 (SNAIL), and Snail family zinc finger 2 (SLUG) mRNA expression levels among the various DF cells cultured in suspension for 120 hours. This PCR reaction was repeated 3 times with 3 technical replicates. (F) TWIST1 mRNA fold change in the various FNE cells. Values are normalized to FNE control cells. All data shown as the median (horizontal bar), interquartile range (box), and minimum/maximum values (whiskers). Statistical analysis performed using 1-way ANOVA (E), or 1-way ANOVA and post-hoc Tukey-Kramer test (F). *P < 0.05 comparing R175H pLKO to control pLKO or p53shRNA.