Figure 3. Δ133p53β isoform promotes invasion in breast cancer cells.

(A) RNA chart of the different isoforms of TP53 in this study showing the exons (boxes) and introns (horizontal lines, not to scale). Alternative promoters are shown as arrows and alternative splices are depicted with the lines above as they connect different exons. Location of the different siRNAs used is indicated below the chart. Below is a list of the p53 isoforms that remain after transfection of the different siRNAs, indicated by a cross. (B) Specific inhibition of some p53 isoforms expression decreases invasiveness. MDA-MB231 D3H2LN cells were transfected with si133-1 or si133-2, two distinct siRNAs specific for the 5’UTR of Δ133p53 mRNAs; or with siTAp53, a siRNA targeting TP53 exon-2 depleting all p53 isoforms except the Δ133p53 (α, β, γ) isoforms; or with siβ, a siRNA targeting the alternatively spliced exon-9β of TP53; or with siNS, a non-specific siRNA used as negative control. (C) ' Rescue' experiments. Re-introductions of si-133–resistant mutant Δ133p53α-R280K, Δ133p53β-R280K or Δ133p53γ-R280K restore the invasive activity in MDA-MB231 D3H2LN cells previously depleted of either Δ133p53 (α, β, γ) isoforms after transfection with si133-1 or si133-2, or β p53 isoforms (p53β, Δ40p53β and Δ133p53β) (n = 4). (D) Inhibition of endogenous p53 protein isoforms induces expression of epithelial features associated with decreased invasiveness. The expression of endogenous p53 protein isoforms after transfection of MDA-MB231 D3H2LN cells with si133-2, siβ, siE7 or control siRNA (siNS) was analyzed by western blotting using pantropic p53 isoforms antibody Sapu or KJC8, a β-specific p53 antibody recognizing p53β, Δ40p53β and Δ133p53β. The expression of two EMT markers (Vimentin and E-Cadherin) was determined in parallel. Ku80 was used as a loading control. * cross-reaction. (E) Quantification of E-Cadherin mRNA in MDA-MB231 D3H2LN cells transfected with siRNA si133-2, siβ, siE7 or siNS (control) used as a negative control. For all RT-qPCR experiments, expression levels were normalized to TBP. Results are expressed relative to TBP mRNA and represent means ± SEMs of N = 4 independent experiments; *p<0.05; **p<0.01 (F) WT ∆133p53β promotes cell invasion. Weakly invasive MCF7 cells were transfected with Δ133p53β expression vector or the empty expression vector (Control). Cells were challenged for their invasive potential after 48 hr. The values are plotted as means ± SEMs of at least 3 independent experiments; *p<0.05.

DOI: http://dx.doi.org/10.7554/eLife.14734.012

Figure 3—figure supplement 1. Δ133p53 (α, β and γ) expression in MCF7 breast cancer cells (related to Figure 3).

(A) Quantitative RT-qPCR (TaqMan) of Δ133p53 (α, β and γ), TAp53 (α, β and γ) or β mRNAs in MDA-MB231 D3H2LN cells transfected with non-specific siRNA (siNS), or p53 specific siRNA si133-1, si133-2, siTAp53 or siβ, respectively. For all RT-qPCR (TaqMan) experiments, expression levels were normalized to TBP. Results are expressed as relative expression to TBP mRNA and represent means ± SEMs of N = 4 independent experiments; **p<0.01. (B) Western blot of ectopically expressed Δ133p53α, Δ133p53β and Δ133p53γ in MDA MB231 D3H2LN breast cancer cells using Sapu p53 pantropic or anti-Flag antibodies after Δ133p53 depletion with si∆133–1 (left panel) or si∆133–2 (right panel). (C) Western blot withSapu p53 pantropic or anti-Flag antibodies of ectopically expressed Δ133p53β in MDA MB231 D3H2LN breast cancer cells previously depleted of all b p53 isoforms with siRNA siβ (D) Expression of ∆133p53 (α, β and γ) mRNA variants in MCF7 cells and in HCT116 cells. Expression was determined by nested RT-PCR (E) Western blot of ectopically expressed Δ133p53β in MCF7 breast cancer cells using Sapu antibody.

DOI: http://dx.doi.org/10.7554/eLife.14734.013