Germain Gillet

Antagonistic regulation of EMT by TIF1γ and Smad4 in mammary epithelial cells

TGF‐β is a potent inducer of epithelial‐to‐mesenchymal transition (EMT), a process involved in tumour invasion. TIF1γ participates in TGF‐β signalling. To understand the role of TIF1γ in TGF‐β signalling and its requirement for EMT, we analysed the TGF‐β1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ‐silenced cells after TGF‐β1 treatment, whereas Smad4 inactivation completely blocked this process. Accordingly, the functions of several TIF1γ target genes can be linked to EMT, as shown by microarray analysis. As a negative regulator of Smad4, TIF1γ could be crucial for the regulation of TGF‐β signalling. Furthermore, TIF1γ binds to and represses the plasminogen activator inhibitor 1 promoter, demonstrating a direct role of TIF1γ in TGF‐β‐dependent gene expression. This study shows the molecular relationship between TIF1γ and Smad4 in TGF‐β signalling and EMT.

Bax-derived membrane-active peptides act as potent and direct inducers of apoptosis in cancer cells

Although many cancer cells are primed for apoptosis, they usually develop resistance to cell death at several levels. Permeabilization of the outer mitochondrial membrane, which is mediated by proapoptotic Bcl-2 family members such as Bax, is considered as a point of no return for initiating apoptotic cell death. This crucial role has placed Bcl-2 family proteins as recurrent targets for anticancer drug development. Here, we propose and demonstrate a new concept based on minimal active versions of Bax to induce cell death independently of endogenous Bcl-2 proteins. We show that membrane-active segments of Bax can directly induce the release of mitochondria-residing apoptogenic factors and commit tumor cells promptly and irreversibly to caspase-dependent apoptosis. On this basis, we designed a peptide encompassing part of the Bax pore-forming domain, which can target mitochondria, induce cytochrome c release and trigger caspase-dependent apoptosis. Moreover, this Bax-derived ‘poropeptide’ produced effective tumor regression after peritumoral injection in a nude mouse xenograft model. Thus, peptides derived from proteins that form pores in the mitochondrial outer membrane represent novel templates for anticancer agents.

Bcl-wav and the mitochondrial calcium uniporter drive gastrula morphogenesis in zebrafish

Bcl-2 proteins are acknowledged as key regulators of programmed cell death. However, increasing data suggest additional roles, including regulation of the cell cycle, metabolism and cytoskeletal dynamics. Here we report the discovery and characterization of a new Bcl-2-related multidomain apoptosis accelerator, Bcl-wav, found in fish and frogs. Genetic and molecular studies in zebrafish indicate that Bcl-wav and the recently identified mitochondrial calcium uniporter (MCU) contribute to the formation of the notochord axis by controlling blastomere convergence and extension movements during gastrulation. Furthermore, we found that Bcl-wav controls intracellular Ca(2+) trafficking by acting on the mitochondrial voltage-dependent anion channel, and possibly on MCU, with direct consequences on actin microfilament dynamics and blastomere migration guidance. Thus, from an evolutionary point of view, the original function of Bcl-2 proteins might have been to contribute in controlling the global positioning system of blastomeres during gastrulation, a critical step in metazoan development.

The Apoptotic Regulator Nrz Controls Cytoskeletal Dynamics via the Regulation of Ca2+ Trafficking in the Zebrafish Blastula

Bcl-2 family members are key regulators of apoptosis. Their involvement in other cellular processes has been so far overlooked. We have studied the role of the Bcl-2 homolog Nrz in the developing zebrafish. Nrz was found to be localized to the yolk syncytial layer, a region containing numerous mitochondria and ER membranes. Nrz knockdown resulted in developmental arrest before gastrulation, due to free Ca(2+) increase in the yolk cell, activating myosin light chain kinase, which led to premature contraction of actin-myosin cables in the margin and separation of the blastomeres from the yolk cell. In the yolk syncytial layer, Nrz appears to prevent the release of Ca(2+) from the endoplasmic reticulum by directly interacting with the IP3R1 Ca(2+) channel. Thus, the Bcl-2 family may participate in early development, not only by controlling apoptosis but also by acting on cytoskeletal dynamics and cell movements via Ca(2+) fluxes inside the embryo.

Oocytes and early embryos selectively express the survival factor BCL2L10

Apoptosis has been reported in oocytes and human preimplantation embryos both in vitro and in vivo. BCL-2 family proteins are likely to play a pivotal role in controlling oocyte and early embryo degeneration. However, no BCL-2-related survival factors have been identified that would specifically function during oocyte maturation, after fertilization and during early embryogenesis. Here, we performed a comprehensive tissue expression pattern analysis of the BCL-2 family at the mRNA level. While expression of various members was detected in human oocytes and during early primate embryogenesis, our data indicate that BCL2L10 is the predominant maternally loaded Bcl-2 family transcript, revealing an evolutionary conserved expression profile at the egg-to-zygote transition. We provide evidence that BCL2L10 is associated with the microtubule binding protein translationally controlled tumor protein and mitochondria, with a stage-specific redistribution along the pericortical regulatory ooplasm. In dying oocytes, BCL2L10 colocalized with proapoptotic BAX and neutralization of BCL2L10 accelerated oocyte death. We propose BCL2L10 as a novel and prime candidate related to oocyte maturation, fertility, and embryo developmental competence.

Nrh, a human homologue of Nr-13 associates with Bcl-Xs and is an inhibitor of apoptosis

In search of human homologues of the anti-apoptotic protein Nr-13, we have characterized a human EST clone that potentially encodes a protein, which is the closest homologue of Nr-13 among the Bcl-2 family members, to date known, in humans. Phylogenetic analyses suggest Human nrh, Mouse diva/boo and Quail nr-13 to be orthologous genes. The nrh gene has the same overall organization as nr-13 and diva/boo with one single intron interrupting the ORF at the level of the Bcl-2-homology domain BH2. RT–PCR-based analysis of nrh expression indicated that this gene is preferentially expressed in the lungs, the liver and the kidneys. Interestingly, two in frame ATG codons can lead potentially to the synthesis of two products, one of them lacking 10 aminoacids at the N-terminal end. Sequence alignment with Nr-13 and Diva/Boo in addition to secondary structure prediction of the nrh transcript suggested that the shortest protein will be preferentially synthetized. Immunohistochemical analyses have revealed that Nrh is associated with mitochondria and the nuclear envelope. Moreover, Nrh preferentially associates with the apoptosis accelerator Bcl-Xs and behaves as an inhibitor of apoptosis both in yeast and vertebrate cells.

Genetic and Pharmacologic Inhibition of mTORC1 Promotes EMT by a TGF-β–Independent Mechanism

Epithelial-to-mesenchymal transition (EMT) is a transdifferentiation process that converts epithelial cells into highly motile mesenchymal cells. This physiologic process occurs largely during embryonic development but is aberrantly reactivated in different pathologic situations, including fibrosis and cancer. We conducted a siRNA screening targeted to the human kinome with the aim of discovering new EMT effectors. With this approach, we have identified mTOR complex 1 (mTORC1), a nutrient sensor that controls protein and lipid synthesis, as a key regulator of epithelial integrity. Using a combination of RNAi and pharmacologic approaches, we report here that inhibition of either mTOR or RPTOR triggers EMT in mammary epithelial cells. This EMT was characterized by the induction of the mesenchymal markers such as fibronectin, vimentin, and PAI-1, together with the repression of epithelial markers such as E-cadherin and ZO-3. In addition, mTORC1 blockade enhanced in vivo migratory properties of mammary cells and induced EMT independent of the TGF-β pathway. Finally, among the transcription factors known to activate EMT, both ZEB1 and ZEB2 were upregulated following mTOR repression. Their increased expression correlated with a marked reduction in miR-200b and miR-200c mRNA levels, two microRNAs known to downregulate ZEB1 and ZEB2 expression. Taken together, our findings unravel a novel function for mTORC1 in maintaining the epithelial phenotype and further indicate that this effect is mediated through the opposite regulation of ZEB1/ZEB2 and miR-200b and miR-200c. Furthermore, these results suggest a plausible etiologic explanation for the progressive pulmonary fibrosis, a frequent adverse condition associated with the therapeutic use of mTOR inhibitors.

Rounding of aggregates of biological cells: Experiments and simulations

The influence of surface tension and size on rounding of cell aggregates is studied using chick embryonic cells and numerical simulations based on the cellular Potts model. Our results show exponential relaxation in both cases as verified in previous studies using 2D Hydra cell aggregates. The relaxation time decreases with higher surface tension as expected from hydrodynamics laws. However, it increases faster than linearly with aggregate size. The results provide an additional support to the validity of the cellular Potts model for non-equilibrium situations and indicate that aggregate shape relaxation is not governed by the hydrodynamics of viscous liquids.

Src tyrosine kinase inhibits apoptosis through the Erk1/2- dependent degradation of the death accelerator Bik

Src, the canonical member of the non-receptor family of tyrosine kinases, is deregulated in numerous cancers, including colon and breast cancers. In addition to its effects on cell proliferation and motility, Src is often considered as an inhibitor of apoptosis, although this remains controversial. Thus, whether the ability of Src to generate malignancies relies on an intrinsic aptitude to inhibit apoptosis or requires preexistent resistance to apoptosis remains somewhat elusive. Here, using mouse fibroblasts transformed with v-Src as a model, we show that the observed Src-dependent resistance to cell death relies on Src ability to inhibit the mitochondrial pathway of apoptosis by specifically increasing the degradation rate of the BH3-only protein Bik. This effect relies on the activation of the Ras–Raf–Mek1/2–Erk1/2 pathway, and on the phosphorylation of Bik on Thr124, driving Bik ubiquitylation on Lys33 and subsequent degradation by the proteasome. Importantly, in a set of human cancer cells with Src-, Kras- or BRAF-dependent activation of Erk1/2, resistances to staurosporine or thapsigargin were also shown to depend on Bik degradation rate via a similar mechanism. These results suggest that Bik could be a rate-limiting factor for apoptosis induction of tumor cells exhibiting deregulated Erk1/2 signaling, which may provide new opportunities for cancer therapies.

Modulation of Nr-13 antideath activity by peptide aptamers.

Tumor cells are characterized by deregulated proliferation and resistance to proapoptotic stimuli. The Bcl-2 family of antiapoptotic proteins is overexpressed in a large number of chemoresistant tumors. Downregulation or inhibition of antiapoptotic proteins might result in the sensitization of cancer cells to chemotherapeutic agents. In the present study, we took advantage of the peptide aptamer strategy to target Nr-13, a Bcl-2 antiapoptotic protein involved in neoplastic transformation by the Rous sarcoma virus. We isolated peptide aptamers that behave as Nr-13 regulators, in vitro and in mammalian cells in culture. Some of these aptamers have potential proapoptotic activities. These data suggest that peptide aptamers targeting the Bcl-2 family of apoptosis inhibitors may be useful for the development of anticancer molecules.