Fig. 6: Reduction of CASP3 expression through SP1 inhibition limits melanoma dissemination potential. | Cell Death & Disease

Fig. 6: Reduction of CASP3 expression through SP1 inhibition limits melanoma dissemination potential.

From: Atypical contribution of caspase-3 to melanoma cancer cell motility by regulation of coronin 1B activity

Fig. 6

A Schematic representation of CASP3 promoter described by Sudhakar et al., 2008, FEBS J. B Analysis of SP1 and CASP3 mRNA expression in melanoma patients with melanoma (R = 0.51; p-value = 9,9 * 10-32). C CASP3 mRNA expression in control and SP1-knockdown WM793 cells, relative to GAPDH. D Western blot analysis of CASP3 and SP1 in control and SP1 siRNA-transfected WM793 cells. HSC70 serves as a loading control. E Analysis of CASP3 expression in WM852 transfected with increasing concentrations of a SP1-expressing plasmid. HSC70 serves as a loading control. F Quantification of the migration potential of parental and SP1-knockdown WM793 cells through wound area measurement (n = 3, one representative experiment shown, two-way ANOVA statistical test; Statistical significance: ns - P > 0.05; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001; **** - P ≤ 0.0001). G DiD-labelled SP1-knockdown and DiO-labeled control WM793 cells were pre-mixed in equal numbers and injected as shown in 3L. A representative epifluorescence image of a whole embryo shows perivitelline homing and caudal blood vessels invasion of cancer cells. White arrows point towards invading cells. H Quantification of invaded metastatic cells per embryo tail vein (data represent mean with SEM, n = 32 embryos from three independent experiments, t-test, **** p < 0.0001). I Analysis of CASP3 and SP1 expression in WM793 treated with increasing doses of mithramycin A (100 nM, 200 nM and 300 nM). GAPDH serves as a loading control. J Wound healing migration assay for WM793 cells treated with 0, 100 or 200 nM of mithramycin A (n = 3, one representative experiment shown, two-way ANOVA statistical test; Statistical significance: ns - P > 0.05; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001; **** - P ≤ 0.0001). K DiD-labelled mithramycin A treated (100 nM) and DiO-labelled control WM793 cells were pre-mixed in equal numbers and injected as shown in 3L. A representative epifluorescence image of a whole embryo shows perivitelline homing and caudal blood vessels invasion of cancer cells. White arrows point towards invading cells. L Quantification of invaded metastatic cells per embryo (data represent mean with SD, n = 29 embryos from three independent experiments, t-test, ****p < 0.0001). M Model: Caspase-3 is highly expressed in melanoma cells, most likely through a transcriptional regulation by SP1 that could be therapeutically blocked by treatment with mithramycin A. Caspase-3 interacts with coronin 1B (CORO1B), promoting its phosphorylation and localization at the cellular leading edge. CORO1B then interacts with ARP2/3 complex at the leading edge contributing to continuous cycles of actin polymerization and depolymerization. Caspase-3 downregulation leads to lower CORO1B phosphorylation, defects in its localization at the leading edge of melanoma cells and therefore decreased cell motility.

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