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 7

TWIST family BHLH transcription factor 1 (TWIST1) modulates phenotypes associated with mutant p53 (m-p53) expression in fallopian tube nonciliated (FNE) cells.

Options: View larger image (or click on image) Download as PowerPoint
TWIST family BHLH transcription factor 1 (TWIST1) modulates phenotypes a...
(A) Western blot of lysates of FNE-p53shRNA cells expressing empty vector or TWIST1 fused to the estrogen receptor (TWIST1-ER). (B) Phase-contrast images (Supplemental Video 11) documenting time-dependent (120 hours) disintegration of cellular clusters composed of FNE-p53shRNA cells transduced with control plasmid (empty) or TWIST1-ER and treated with 25 nM 4-hydroxytamoxifen 5 days prior to data acquisition. Scale bar: 150 μm. Eight to ten movies were recorded per condition during 1 acquisition session with 100–150 cells imaged per cluster per movie. (C) Phase-contrast and pseudocolored images of ethidium bromide (EtBr) incorporation into FNE-m-p53R175H cells expressing control plasmid or TWIST1-ER. Scale bar: 100 μm. (D) Quantification of EtBr incorporation into the various FNE cell lines. n = 35 (FNE-p53shRNA and control) and n = 55 (FNE-p53shRNA-TWIST1-ER) clusters scored per condition in 2 separate experiments. (E) Phase-contrast and pseudocolored images of EtBr incorporation into FNE-m-p53R175H cells transduced with empty pLKO or TWIST1 shRNAs. Scale bar: 100 μm. (F) Quantification of EtBr incorporation into the various FNE cell lines. n = 18–20 clusters scored per condition in 2 separate experiments. All data shown as the median (horizontal bar), interquartile range (box), and minimum/maximum values (whiskers). Statistical analysis performed using 2-tailed Student’s t test (D) and 1-way ANOVA and post-hoc Tukey-Kramer test (F). *P < 0.05 comparing sh#1 or sh#2 to pLKO.