TAp73β-mediated suppression of cell migration requires p57Kip2 control of actin cytoskeleton dynamics

Abstract

The TP73 gene, a member of the p53 family, due to the use of different promoters and alternative splicing, is transcribed into different isoforms with contrasting attributes and which contribute to its functional diversity. Considerable efforts are made to identify the functional diversity of the p73 splicing variants during tumorigenesis. TAp73α and TAp73β isoforms have been shown to differentially regulate cell cycle progression, differentiation and apoptosis. Interestingly, a particular increase in expression of the TAp73 isoform, in favor of the α splicing variant, has been reported in multiple tumour types. Here, we report a distinctive role for TAp73β isoform in the control of cell migration and invasion. In fact, TAp73β- dependent induction of p57(Kip2) expression accounted for inhibitory effects on the actin cytoskeleton dynamics and thereby cancer cell motility. In contrast, TAp73α is not able to induce p57(Kip2) expression, and exhibits a positive effect on actin cytoskeleton dynamics as well as cell migration and invasion. In conclusion, the inhibitory effect on cell migration and invasion of TAp73β would qualify this distinct p73 isoform as tumor suppressor gene. In contrast, the promoting effect of TAp73α on cell motility and invasion strengthens the potential oncogenic activities of this p73 isoform.

Figure 1. p73β, but not p73α expression, inhibits cell migration

Human cervical cancer HeLa cells were transfected with expression vector encoding for p73α or p73β, and compared with cells that were transfected with the backbone pcDNA3 expression vector. Protein expression was confirmed by immunoblotting against p73α/β, using G3PDH as loading control (A). Cell motility was determined by wound-healing assay measuring cell migration into the wound (B). Data shows mean values of three independent experiments +SEM (C). At 6 hours, statistical analysis of p73α versus p73β indicates a p-value ** <0.01.

Figure 2. p73β expression reduces cell invasive capacity

The invasive ability of p73α- and p73β-expressing HeLa cells as compared to control pcDNA3 transfected HeLa cells were determined in real-time during 42 h, using Matrigel-coated transwell chamber and the xCELLigence RTCA DP analyzer. Cell Index represents the degree of cell invasion into the reconstituted basement membrane matrix Matrigel. Data shows representative invasion curves for each condition performed in triplicate +SD, out of three independent experiments. At 24 hours, statistical analysis of p73α versus p73β indicates a p-value * <0.05.

Figure 3. Actin cytoskeleton dynamics are impaired in p73β-expressing cells

HeLa cells were transfected with expression vector encoding for p73α or p73β, and compared with control cells transfected with the corresponding empty vector. The mobile actin fraction in p73α-, or p73β-expressing HeLa cells was measured by FRAP analysis of cells co-transfected with actin-GFP to visualize the actin cytoskeleton. Values represent the % fluorescence recovery over time of actin-GFP after bleaching. Arrows indicate the photobleached area (A). For every FRAP experiment 3-7 cells per condition were used. Average of three independent experiments is presented in the figure (B).

Figure 4. p73β regulation of the actin cytoskeleton dynamics is dependent on induction of p57^Kip2

HeLa cells were co-transfected with the expressing vector encoding for p73β, together with a p57^Kip2 siRNA or a scrambled sequence siRNA. p57^kip2 expression and knockdown was confirmed by immunoblotting against p57, using G3PDH as a loading control. HeLa cells co-transfected with the expressing vector encoding for p73α, p57^Kip2 or the backbone expression vector together with the scrambled sequence siRNA were used as controls. (A). FRAP assay on GFP-actin-expressing cells was performed to evaluate actin cytoskeleton dynamincs. Bleached area of actin-GFP is indicated by an arrow (B). The actin turnover is measured as % recovery of the actin-GFP signal during 150 s (C). For every FRAP experiment 4-7 cells per condition were used. Average of three independent experiments is presented in the figure (C).