Mutant p53 regulates ovarian cancer transformed phenotypes through autocrine matrix deposition
Marcin P. Iwanicki, Hsing-Yu Chen, Claudia Iavarone, Ioannis K. Zervantonakis, Taru Muranen, Marián Novak, Tan A. Ince, Ronny Drapkin, Joan S. Brugge
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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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 4

shRNA-mediated attenuation of wild-type p53 does not induce phenotypes associated with mutant p53 (m-p53) overexpression.

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shRNA-mediated attenuation of wild-type p53 does not induce phenotypes a...
(A) Representative phase-contrast video clips of fallopian tube nonciliated epithelial (FNE) cells transduced with empty vector (pLKO), p53shRNA#1, p53shRNA#2, or m-p53R175H plus empty pLKO (Supplemental Video 7) and cultured in suspension for the indicated time points. Arrows point to dying cells. Clips are representative of 10 movies per condition with 100–150 cells imaged per movie, acquired in 1 recording session. (B) Phase-contrast and pseudocolored fluorescence images representing the degree of ethidium bromide (EtBr; red) incorporation into cellular clusters (green). (C) Quantification of the distribution of EtBr incorporation among the various FNE suspended cell clusters. Three independent experiments performed with n = 39 (control), n = 33 (p53shRNA#1), n = 29 (p53shRNA#2), and n = 33 (m-p53R175H) cellular clusters analyzed. (D) Representative differential interference contrast (DIC) and pseudocolored (red) fluorescence images demonstrating the ability of control (pLKO), p53shRNA#1, p53shRNA#2, and m-p53R175H FNE cell clusters to clear the mesothelium (mesothelial cells expressing red fluorescent protein [RFP]), 8 hours after seeding. (E) Quantification of the mesothelial clearance distribution by the various FNE cell lines from a representative experiment. This experiment was repeated twice with n = 7–8 cellular clusters analyzed per condition. All data shown as the median (horizontal bar), interquartile range (box), and minimum/maximum values (whiskers). Statistical analysis performed using 1-way ANOVA and post-hoc Tukey-Kramer tests comparing m-p53R175H with each group (control pLKO, p53sh#1, and p53sh#2). *P < 0.05. Scale bar: 150 μm.