Snail blocks the cell cycle and confers resistance to cell death

Abstract

The Snail zinc-finger transcription factors trigger epithelial-mesenchymal transitions (EMTs), endowing epithelial cells with migratory and invasive properties during both embryonic development and tumor progression. During EMT, Snail provokes the loss of epithelial markers, as well as changes in cell shape and the expression of mesenchymal markers. Here, we show that in addition to inducing dramatic phenotypic alterations, Snail attenuates the cell cycle and confers resistance to cell death induced by the withdrawal of survival factors and by pro-apoptotic signals. Hence, Snail favors changes in cell shape versus cell division, indicating that with respect to oncogenesis, although a deregulation/increase in proliferation is crucial for tumor formation and growth, this may not be so for tumor malignization. Finally, the resistance to cell death conferred by Snail provides a selective advantage to embryonic cells to migrate and colonize distant territories, and to malignant cells to separate from the primary tumor, invade, and form metastasis.

Figure 1.

Snail expression impairs cell proliferation. (A) BrdU incorporation after a 1-h pulse in MDCK cells stably transfected with Snail (MDCK-Snail) or the empty vector (MDCK-Mock). Bright-field images after 24 h in culture. (B) FACS analysis of the cell cycle in MDCK-Mock and MDCK-Snail cells after different times in culture.

Figure 2.

Snail alters the expression of proteins involved in progression from G1 to S phase. Western blot analysis of MDCK-Mock and MDCK-Snail cells. (A) Levels of the G1 checkpoint molecules, the cdk inhibitors p21 and p27, and the degree of Rb phosphorylation. (B) Levels of p21 after different times in culture. Rb, retinoblastoma protein; pRb, hypophosphorylated state; ppRb, hyperphosphorylated state. Representative experiments are shown (n = 4).

Figure 3.

Snail represses Cyclin D2 transcription. (A) Analysis of D cyclins and their partner cdk4. Immunoblotting of total erk2 was used as a control of gel loading. (B) Analysis of Cyclins D1 and D2 transcription by Northern blot of RNA extracted from MDCK-Mock and MDCK-Snail cells after different times in culture. The GAPDH probe was used as a control of loading. (C) Activity of the Cyclin D2 promoter. Luciferase reporter constructs carrying the wild-type human Cyclin D2 promoter (–1624) or independent deletions/mutations in the two E-boxes were transfected into MCA3D cells together with a mouse Snail expression vector or the empty vector (pcDNA3) as a control. Luciferase activity was assayed 40 h after transfection. Activity is expressed relative to that of the wild-type construct. Results are the mean values ± S.E. of duplicates from four independent experiments.

Figure 4.

An inverse correlation exists between Cyclin D2 and Snail expression in mouse embryos. Whole-mount in situ hybridization of 8.5-dpc mouse embryos (A–C) and transverse paraffin sections of the same embryos taken at the level of the posterior hindbrain (D–F), the trunk (G–I), and the allantois (J–L). Snail expression can be observed at the edges of the neural plate (D, pnc) corresponding to premigratory crest cells undergoing EMT. Snail expression is maintained in crest cells after delamination (G, nc), and it is also apparent in the decondensing somites (G, s) and in the allantois (J, al). An inverse correlation between Snail and Cyclin D2 transcripts is readily observed in all the tissues analyzed (cf. D,G,J and F,I,L). (K) Although this correlation is not so striking for Snail and Cyclin D1, note that Cyclin D1 expression is not detected in regions with high levels of Snail transcripts such as the allantois. (al) Allantois; (hb) hindbrain; (nc) neural crest; (np) neural plate; (nt) neural tube; (pnc) premigratory neural crest; (s) somite.

Figure 5.

An inverse correlation exists between proliferation and Snail expression in mouse embryos. The embryos in A and B show a side-by-side comparison between Snail expression and BrdU incorporation as a measure of cell proliferation in the whole embryo in culture. An overall complementary pattern is observed, that can be better examined in the sections taken at the level of the forebrain (C,D) and the base of the allantois (E,F). G and H show sections at the trunk level to compare Snail expression with histone H3 phosphorylation, taken as a measure of cells undergoing mitosis. The squares mark the Snail-expressing region of the neural epithelium. (a) Amnion; (al) allantois; (fb) forebrain; (h) heart; (hb) hindbrain.

Figure 6.

Snail confers resistance to apoptosis induced by serum deprivation. (A) Cell viability was assessed by propidium iodide staining 48 h after serum depletion. (B) Caspase-3 activity at different times after serum removal represented as mean values ± S.E. from three independent experiments carried out with duplicate dishes. Note the low levels of activity in Snail-expressing cells 48h after serum deprivation compared to the mock-transfected cells. (C) Cell death visualized using Nile Blue Sulphate staining is compared side-by-side with Snail expression in the head of an 8.5-dpc mouse embryo. The pattern of cell death assessed by Nile Blue Sulphate (NBS) staining (stars) is complementary to that of Snail (brackets). A similar embryo hybridized with Krox-20 to indicate the relative position of pre-rhombomeres (pr) 3 and 5 in the hindbrain to help compare the pattern of cell death and Snail expression. The inverse correlation can be better assessed in the high-power photographs. (fb) Forebrain; (h) heart; (mb) midbrain; (sc) anterior spinal cord.

Figure 7.

Snail activates survival pathways. Molecules from different survival pathways were analyzed in cells cultured in the absence of serum and collected at different times. The levels of active ERKs (phospho-erk1 and phospho-erk2; A), active Akt (B), and Bcl-x_L (C) were analyzed by Western blot and found to be increased in Snail-expressing cells. Total erk2 was used as a control for gel loading. (B) Higher levels of PI3K activity were also detected in Snail-expressing cells as analyzed by thin-layer chromatography, compatible with the higher levels of phosphorylation found for Akt.

Figure 8.

Snail confers resistance to the cell death induced by TNF-α. Mock and MDCK-Snail-expressing cells were treated with TNF-α (5 ng/mL) after being pretreated with cycloheximide (0.5 μg/mL for 30 min) to prevent the induction of the survival protein NFκB. (A) Photographs of the cultures taken after 16 or 24 h of treatment. (B,C) The activity of the death receptors-specific caspase-8 and effector caspase-3, respectively, are shown from one representative experiment. Note the low activity of both caspases in Snail-expressing cells, explaining the healthy appearance observed in A.

Figure 9.

Slug expression correlates with little proliferation and protects the developing neural tube from physiological cell death in the chick. (A) In ovo BrdU incorporation was analyzed in transverse sections. One such section from the trunk of a stage 11 chicken embryo is shown. (B) A similar section hybridized with a Slug probe. Note the absence of BrdU in the premigratory neural crest, showing high levels of Slug expression. (C,D) The pattern of cell death in the hindbrain region of a stage 12 chick embryo as assessed by NBS and TUNEL staining, respectively. Compare the pattern of cell death (blue and brown stars in C,D) with that of Slug transcripts (E). As previously described, r4 shows very few apoptotic cells, coinciding with high levels of Slug transcripts (brackets). (F–H) An embryo electroporated with plasmids containing chick Slug and GFP cDNAs at stage 8 and analyzed 15 h later (stage 12). (F) GFP (and thus, Slug) expression is observed in the right-hand side of the neural tube and in cells migrating from it. (G) NBS staining of the same embryo shows a striking decrease in cell death in the side where Slug is overexpressed. (H) A higher-magnification picture that allows a better assessment of the region protected from cell death. The dotted lines demarcate the borders of the neural tube and the otic vesicle. The black stars indicate a region of ectodermal cell death that appears symmetrical on both sides of the embryo (see text). (hb) Hindbrain; (mb) midbrain; (nc) neural crest; (nt) neural tube; (ov) otic vesicle, (s) somite.