Snail Family Members Unequally Trigger EMT and Thereby Differ in Their Ability to Promote the Neoplastic Transformation of Mammary Epithelial Cells

<div><p>By fostering cell commitment to the epithelial-to-mesenchymal transition (EMT), SNAIL proteins endow cells with motility, thereby favoring the metastatic spread of tumor cells. Whether the phenotypic change additionally facilitates tumor initiation has never been addressed. Here we demonstrate that when a SNAIL protein is ectopically produced in non-transformed mammary epithelial cells, the cells are protected from anoikis and proliferate under low-adherence conditions: a hallmark of cancer cells. The three SNAIL proteins show unequal oncogenic potential, strictly correlating with their ability to promote EMT. SNAIL3 especially behaves as a poor EMT-inducer comforting the concept that the transcription factor functionally diverges from its two related proteins.</p></div

SNAIL proteins promote MCF10A commitment to EMT.

<p>MCF10A cells were infected with constructs encoding SNAIL proteins as indicated at the top and characterized. (A) Representative photomicrographs of cells obtained by phase contrast microscopy. (B) Analysis of epithelial (E-caherin, β-catenin) and mesenchymal (fibronectin, vimentin) markers by immunofluorescence. (C) Analysis by flow cytometry of CD44 and CD24. (D) Invasion assay. Percentages of invasive cells are indicated. (E) Upper panels: analysis of ectopic <i>SNAI</i> expression by qRT-PCR. Transcript levels are expressed with respect to transcripts of the <i>HPRT1</i> housekeeping gene. Lower panels: western blot analysis of SNAIL proteins. Proteins of interest are indicated with stars. (F) Analysis of epithelial and mesenchymal markers by western blotting.</p

SNAIL proteins confer a survival advantage to MCF10A cells under low-adherence conditions.

<p>(A) MCF10A cells infected with SNAIL-protein-encoding constructs, as indicated at the top, were cultured in ultra-low attachment dishes for different periods of time as indicated on the right. Cells were then stained with annexin V-FITC and propidium iodide and analyzed by flow cytometry. The results shown are representative of three independent experiments. (B) Histogram showing percentages of apoptotic cells (including annexin V⁺/PI⁻ and annexin V⁺/PI⁺ cells) means with SD of triplicate experiments. (<b>C</b>) Analysis of cleaved caspase-3 fragment by western blotting.</p

SNAIL proteins confer a survival advantage to HMEC-hTERT cells under low-adherence conditions.

<p>(A) Upper panels: HMEC-derived cell lines were cultured in ultra-low attachment dishes for different periods of time as indicated on the right. The cells were then stained with annexin V-FITC and propidium iodide and analyzed by flow cytometry. (B) Percentages of apoptotic cells (including Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺ cells) are indicated as means ±SD of triplicate experiments. (C) Analysis of the cleaved caspase-3 fragment by western blotting. (D) Examination by western blotting of the status of the ERK and AKT pathways. P-ERK and P-AKT stand for phospho-T202, Y204 ERK1/2 and phospho-S473 AKT respectively. (E) Expression analysis of <i>ZEB1</i> and <i>ZEB2</i> in HMEC-hTERT cells ectopically expressing either <i>SNAI1</i>, <i>SNAI2</i> or <i>SNAI3</i>. Levels expressed relatively to the housekeeping <i>HPRT1</i> gene transcripts were normalized with respect to HMEC-hTERT cells ±SD of triplicates.</p

Determination by RT-qPCR of <i>SNAI1, SNAI2 and SNAI3</i> transcript levels in human primary mammary tumors.

<p>Levels expressed relatively to housekeeping gene transcripts were normalized with respect to HMEC-hTERT cells. ER/PR: expression analysis of the estrogen and progesterone receptor, + means >10% expressing cells. HER2^+/−: amplification status of the <i>ERBB2</i> gene. SBR: Scarff-Bloom-Richardson grade. Metaplastic tumors include malpighian and sarcomatoïd carcinomas.</p

Twist1 promotes tumor progression <i>in vivo</i>.

<p>Transgene expression was driven by the MMTV-Cre. While MMTV-Cre; <i>Twist1</i> mice (TWIST1; n = 71) do not develop skin lesions, MMTV-Cre;<i>K-rasG12D</i> animals (RAS; n = 89) spontaneously develop anal and oral papillomas. In MMTV-Cre;<i>K-rasG12D</i>;<i>Twist1</i> mice (RAS+TWIST1; n = 19) papillomas evolve into squamous carcinomas. (A) HPS staining of skins either from <i>K-rasG12D</i> or <i>K-rasG12D</i>;<i>Twist1</i> transgenic mice. Transgene expression is induced by the MMTV-Cre. (B) Kaplan-Meier survival curves of transgenic mice. Survival corresponds to the end point of the experiment, the tumor burden requiring euthanasia of the animal. Open circles indicate mice censored from the dataset.</p

Combined expression of H-RAS^G12V and EMT–inducing transcription factors provides HMEC cells with a stem cell–like antigenic phenotype.

<p>Analysis by flow cytometry of the expression of CD44, CD24 (A) and EpCAM (B). Solid blue lines represent staining with FITC-anti-EpCAM antibody and dotted red lines represent staining with FITC-conjugated isotype control. B.D.L.: Below Detection Limit. The claudin-low mesenchymal breast cancer cell line MDA-MB231 was used as a negative EpCAM staining control.</p

Assessment of PP2A activity in hTERT-HMEC derivatives.

<p>PP2A phosphatase activity was assessed and normalized with respect to the parental hTERT-HMEC cells. Activities are expressed in arbitrary units ± SD of triplicate experiments. hTERT-HMECs transduced with SV40 T/t antigens and H-RAS^G12V (HMLER, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002723#pgen.1002723-Elenbaas1" target="_blank">[29]</a>) were used as an internal positive control.</p

EMT induction by embryonic transcription factors facilitates HMEC transformation by an activated form of RAS.

<p>Sequentially, HMECs were immortalized with hTERT (control) and infected with retroviral expression constructs for TWIST1, ZEB1, or ZEB2 and H-RAS^G12V (as depicted on top). (A) Representative photomicrographs of cells obtained by phase contrast microscopy. (B) E-cadherin expression analysis by immunofluorescence. (C) Cell morphology in 3D culture. (D) Transformation potential analysis by means of a soft agar colony formation assay. Numbers of colonies are indicated ± SD of three replicates. (E) Mammosphere formation assay under low adherence conditions. Numbers of mammospheres per 20 000 cells are indicated ± SD of three replicates. (F–G) Expression analysis of TWIST1, ZEB1, ZEB2, H-RAS, and of epithelial and mesenchymal markers by western blotting. Quantification of E-cadherin, vimentin and fibronectin signals, normalized with respect to β-actin expression and HMEC-hTERT-RAS derivatives is shown.</p

Characterization of epithelial and mesenchymal hTERT-shp53/RAS HMEC-derivative subpopulations.

<p>(A, B) Migratory and invasive properties analyses as assessed by Boyden chamber migration and Matrigel invasion assays, respectively. (C) Cellular morphology in 3D culture. (D) Transformation potential analysis assessed by a soft agar colony formation assay. Numbers of colonies are indicated ± SD of triplicate counting. (E) Tumorigenic potential analysis assessed by homotopically xenografts in <i>nude</i> mice. Numbers of mice developing tumors are indicated. (F) Tumor histology: Hematoxylin-Phloxin Saffranin staining (HPS) and cytokeratin, E-cadherin and vimentin expression analysis assessed by immunohistochemistry.</p