EMT Inducers Catalyze Malignant Transformation of Mammary Epithelial Cells and Drive Tumorigenesis towards Claudin-Low Tumors in Transgenic Mice

The epithelial-mesenchymal transition (EMT) is an embryonic transdifferentiation process consisting of conversion of polarized epithelial cells to motile mesenchymal ones. EMT–inducing transcription factors are aberrantly expressed in multiple tumor types and are known to favor the metastatic dissemination process. Supporting oncogenic activity within primary lesions, the TWIST and ZEB proteins can prevent cells from undergoing oncogene-induced senescence and apoptosis by abolishing both p53- and RB-dependent pathways. Here we show that they also downregulate PP2A phosphatase activity and efficiently cooperate with an oncogenic version of H-RAS in malignant transformation of human mammary epithelial cells. Thus, by down-regulating crucial tumor suppressor functions, EMT inducers make cells particularly prone to malignant conversion. Importantly, by analyzing transformed cells generated in vitro and by characterizing novel transgenic mouse models, we further demonstrate that cooperation between an EMT inducer and an active form of RAS is sufficient to trigger transformation of mammary epithelial cells into malignant cells exhibiting all the characteristic features of claudin-low tumors, including low expression of tight and adherens junction genes, EMT traits, and stem cell–like characteristics. Claudin-low tumors are believed to be the most primitive breast malignancies, having arisen through transformation of an early epithelial precursor with inherent stemness properties and metaplastic features. Challenging this prevailing view, we propose that these aggressive tumors arise from cells committed to luminal differentiation, through a process driven by EMT inducers and combining malignant transformation and transdifferentiation

Combined nanomedicines targeting colorectal cancer stem cells and cancer cells.

The study aims to combine the delivery of two anticancer drugs to target both proliferating cancer cells and dormant cancer stem cells (CSCs) present in colorectal cancer. Two drugs were selected and encapsulated in lipid nanocapsules: SN38, the active form of irinotecan, which is unstable in the plasma but active against replicating cells, and salinomycin, a highly toxic ionophore active against cancer stem cells that is not suitable for clinical use. Using an engineered medium that enhanced the ratio of CSCs in HCT116 cell cultures, we demonstrated by clonogenicity tests and in sphere assays that Salinomycin acts mainly on CSCs, while SN38 acts mainly on proliferating cancer cells. In a preclinical murine CRC model, encapsulation of both drugs in lipid nanocapsules reduced their toxicity, including hemolysis, and led to a higher survival than what was observed following treatment with single drugs or non-encapsulated drugs. Nanoparticles loaded with an anticancer drug and salinomycin were effective against the therapy-resistant dormant CSCs and cancer cells

Enforcing local DNA kinks by sequence-selective trisintercalating oligopeptides of a tricationic porphyrin. A polarizable Molecular Dynamics study.

Bisacridinyl-bisarginyl porphyrin (BABAP) is a trisintercalating derivative of a tricationic porphyrin, formerly designed and synthesized in order to selectively target and photosensitize the ten-base pair palindromic sequence d(CGGGCGCCCG)2. We resorted to the previously derived (Far et al., 2004) lowest energy-minimized (EM) structure of the BABAP complex with this sequence as a starting point. We performed polarizable molecular dynamics (MD) on this complex. It showed, over a 150 ns duration, the persistent binding of the Arg side-chain on each BABAP arm to the two G bases upstream from the central porphyrin intercalation site. We subsequently performed progressive shortenings of the connector chain linking the Arg-Gly backbone to the acridine, from n=6 methylenes to 4, followed by removal of the Gly backbone and further connector shortenings, from n=4 to n=1. These resulted into progressive deformations (kinks) of the DNA backbone. In its most accented kinked structure, the DNA backbone was found to have a close overlap with that of DNA bound to Cre recombinase, with, at the level of one acridine intercalation site, negative roll and positive tilt values consistent with those experimentally found for this DNA at its own kinked dinucleotide sequence. Thus, in addition to their photosensitizing properties, some BABAP derivatives could induce sequence-selective, controlled DNA deformations, which are targets for cleavage by endonucleases or for repair enzymes

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 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 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 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

EMT Transcription Factor ZEB1 Represses the Mutagenic POLθ-Mediated End-Joining Pathway in Breast Cancers

International audienceA characteristic of cancer development is the acquisition of genomic instability, which results from the inaccurate repair of DNA damage. Among double-strand break repair mechanisms induced by oncogenic stress, the highly mutagenic thetamediated end-joining (TMEJ) pathway, which requires DNA polymerase theta (POLq) encoded by the POLQ gene, has been shown to be overexpressed in several human cancers. However, little is known regarding the regulatory mechanisms of TMEJ and the consequence of its dysregulation. In this study, we combined a bioinformatics approach exploring both Molecular Taxonomy of Breast Cancer International Consortium and The Cancer Genome Atlas databases with CRISPR/Cas9-mediated depletion of the zinc finger E-box binding homeobox 1 (ZEB1) in claudin-low tumor cells or forced expression of ZEB1 in basal-like tumor cells, two triple-negative breast cancer (TNBC) subtypes, to demonstrate that ZEB1 represses POLQ expression. ZEB1, a master epithelial-to-mesenchymal transition-inducing transcription factor, interacted directly with the POLQ promoter. Moreover, downregulation of POLQ by ZEB1 fostered micronuclei formation in TNBC tumor cell lines. Consequently, ZEB1 expression prevented TMEJ activity, with a major impact on genome integrity. In conclusion, we showed that ZEB1 directly inhibits the expression of POLQ and, therefore, TMEJ activity, controlling both stability and integrity of breast cancer cell genomes. Significance: These findings uncover an original mechanism of TMEJ regulation, highlighting ZEB1 as a key player in genome stability during cancer progression via its repression of POLQ. See related commentary by Carvajal-Maldonado and Wood, p

A stemness-related ZEB1–MSRB3 axis governs cellular pliancy and breast cancer genome stability

International audienceChromosomal instability (CIN), a feature of most adult neoplasms from their early stages onward, is a driver of tumorigenesis. However, several malignancy subtypes, including some triple-negative breast cancers, display a paucity of genomic aberrations, thus suggesting that tumor development may occur in the absence of CIN. Here we show that the differentiation status of normal human mammary epithelial cells dictates cell behavior after an oncogenic event and predetermines the genetic routes toward malignancy. Whereas oncogene induction in differentiated cells induces massive DNA damage, mammary stem cells are resistant, owing to a preemptive program driven by the transcription factor ZEB1 and the methionine sulfoxide reductase MSRB3. The prevention of oncogene-induced DNA damage precludes induction of the oncosuppressive p53-dependent DNA-damage response, thereby increasing stem cells' intrinsic susceptibility to malignant transformation. In accord with this model, a subclass of breast neoplasms exhibit unique pathological features, including high ZEB1 expression, a low frequency of TP53 mutations and low CIN