Targeting cholesterol impairs cell invasion of all breast cancer types.

peer reviewed[en] BACKGROUND: Breast cancer clinical outcome relies on its intrinsic molecular subtype and mortality is almost exclusively due to metastasis, whose mechanism remains unclear. We recently revealed the specific contribution of plasma membrane cholesterol to the invasion of malignant MCF10CAIa but not premalignant MCF10AT and normal MCF10A cell lines in 2D, through invadopodia formation and extracellular matrix (ECM) degradation. In the present study, we address the impact of breast cancer subtypes, mutations and aggressiveness on cholesterol implication in breast cancer cell invasion and 3D spheroid invasion and growth. METHODS: We used nine breast cancer cell lines grouped in four subtypes matching breast tumor classification. Four of these cell lines were also used to generate 3D spheroids. These cell lines were compared for cell invasion in 2D and 3D, spheroid growth in 3D, gelatin degradation, cortactin expression, activation and subcellular distribution as well as cell surface cholesterol distribution and lipid droplets. The effect of plasma membrane cholesterol depletion on all these parameters was determined in parallel and systematically compared with the impact of global matrix metalloproteinase (MMP) inhibition. RESULTS: The six invasive cell lines in 2D were sensitive to partial cholesterol depletion, independently of their subtype, aggressiveness or mutation. Nevertheless, the effect was stronger in the three cell lines able to degrade gelatin. 3D spheroid invasion was also reduced after cholesterol depletion in all breast cancer subtypes tested. Notably, targeting cholesterol was more powerful than MMP inhibition in reducing invasion in both 2D and 3D culture models. Moreover, cholesterol depletion in the six invasive cell lines impaired cortactin distribution in the perinuclear region where invadopodia localized. Breast cancer cell line aggressiveness relied on cholesterol-enriched domains at the ECM-free side and intracellular lipid droplets. Furthermore, the three gelatin-degrading cell lines were characterized by increased cholesterol-enriched submicrometric domains at their ECM-contact side. CONCLUSION: Together, our data suggest cell surface cholesterol combined with lipid droplet labeling as a breast cancer cell aggressiveness marker. They also open the way to test other cholesterol-targeting drugs in more complex models to further evaluate whether cholesterol could represent a strategy in breast cancer therapy

Mechanism of primitive duct formation in the pancreas and submandibular glands: a role for SDF-1

BACKGROUND: The exocrine pancreas is composed of a branched network of ducts connected to acini. They are lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesoderm-derived mesenchyme. The morphogenic mechanisms by which the ductal network is established as well as the signaling pathways involved in this process are poorly understood. RESULTS: By morphological analyzis of wild-type and mutant mouse embryos and using cultured embryonic explants we investigated how epithelial morphogenesis takes place and is regulated by chemokine signaling. Pancreas ontogenesis displayed a sequence of two opposite epithelial transitions. During the first transition, the monolayered and polarized endodermal cells give rise to tissue buds composed of a mass of non polarized epithelial cells. During the second transition the buds reorganize into branched and polarized epithelial monolayers that further differentiate into tubulo-acinar glands. We found that the second epithelial transition is controlled by the chemokine Stromal cell-Derived Factor (SDF)-1. The latter is expressed by the mesenchyme, whereas its receptor CXCR4 is expressed by the epithelium. Reorganization of cultured pancreatic buds into monolayered epithelia was blocked in the presence of AMD3100, a SDF-1 antagonist. Analyzis of sdf1 and cxcr4 knockout embryos at the stage of the second epithelial transition revealed transient defective morphogenesis of the ventral and dorsal pancreas. Reorganization of a globular mass of epithelial cells in polarized monolayers is also observed during submandibular glands development. We found that SDF-1 and CXCR4 are expressed in this organ and that AMD3100 treatment of submandibular gland explants blocks its branching morphogenesis. CONCLUSION: In conclusion, our data show that the primitive pancreatic ductal network, which is lined by a monolayered and polarized epithelium, forms by remodeling of a globular mass of non polarized epithelial cells. Our data also suggest that SDF-1 controls the branching morphogenesis of several exocrine tissues.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Variability of extracellular vesicle release during storage of red blood cell concentrates is associated with differential membrane alterations, including loss of cholesterol-enriched domains

Transfusion of red blood cell concentrates is the most common medical procedure to treat anaemia. However, their storage is associated with development of storage lesions, including the release of extracellular vesicles. These vesicles affect in vivo viability and functionality of transfused red blood cells and appear responsible for adverse post-transfusional complications. However, the biogenesis and release mechanisms are not fully understood. We here addressed this issue by comparing the kinetics and extents of extracellular vesicle release as well as red blood cell metabolic, oxidative and membrane alterations upon storage in 38 concentrates. We showed that extracellular vesicle abundance increased exponentially during storage. The 38 concentrates contained on average 7 × 1012 extracellular vesicles at 6 weeks (w) but displayed a ∼40-fold variability. These concentrates were subsequently classified into 3 cohorts based on their vesiculation rate. The variability in extracellular vesicle release was not associated with a differential red blood cell ATP content or with increased oxidative stress (in the form of reactive oxygen species, methaemoglobin and band3 integrity) but rather with red blood cell membrane modifications, i.e., cytoskeleton membrane occupancy, lateral heterogeneity in lipid domains and transversal asymmetry. Indeed, no changes were noticed in the low vesiculation group until 6w while the medium and the high vesiculation groups exhibited a decrease in spectrin membrane occupancy between 3 and 6w and an increase of sphingomyelin-enriched domain abundance from 5w and of phosphatidylserine surface exposure from 8w. Moreover, each vesiculation group showed a decrease of cholesterol-enriched domains associated with a cholesterol content increase in extracellular vesicles but at different storage time points. This observation suggested that cholesterol-enriched domains could represent a starting point for vesiculation. Altogether, our data reveal for the first time that the differential extent of extracellular vesicle release in red blood cell concentrates did not simply result from preparation method, storage conditions or technical issues but was linked to membrane alterations

MarvelD3 couples tight junctions to the MEKK1-JNK pathway to regulate cell behavior and survival

MarvelD3 is a transmembrane component of tight junctions, but there is little evidence for a direct involvement in the junctional permeability barrier. Tight junctions also regulate signaling mechanisms that guide cell proliferation; however, the transmembrane components that link the junction to such signaling pathways are not well understood. In this paper, we show that MarvelD3 is a dynamic junctional regulator of the MEKK1-c-Jun NH2-terminal kinase (JNK) pathway. Loss of MarvelD3 expression in differentiating Caco-2 cells resulted in increased cell migration and proliferation, whereas reexpression in a metastatic tumor cell line inhibited migration, proliferation, and in vivo tumor formation. Expression levels of MarvelD3 inversely correlated with JNK activity, as MarvelD3 recruited MEKK1 to junctions, leading to down-regulation of JNK phosphorylation and inhibition of JNK-regulated transcriptional mechanisms. Interplay between MarvelD3 internalization and JNK activation tuned activation of MEKK1 during osmotic stress, leading to junction dissociation and cell death in MarvelD3-depleted cells. MarvelD3 thus couples tight junctions to the MEKK1-JNK pathway to regulate cell behavior and survival

Transforming Growth Factor β-Independent Shuttling of Smad4 between the Cytoplasm and Nucleus

Smad4 plays a pivotal role in all transforming growth factor β (TGF-β) signaling pathways. Here we describe six widely expressed alternatively spliced variants of human Smad4 with deletions of different exons in the linker, the region of Smad4 that separates the two well-conserved MH1 and MH2 domains. All these Smad4 variants form complexes with activated Smad2 and Smad3 and are incorporated into DNA-binding complexes with the transcription factor Fast-1, regardless of the amount of linker they contain. However, sequences encoded by exons 5 to 7 in the linker are essential for transcriptional activation. Most importantly, our observation that different Smad4 isoforms have different subcellular localizations has led us to the identification of a functional CRM1-dependent nuclear export signal in the Smad4 linker and a constitutively active nuclear localization signal in the N-terminal MH1 domain. In the absence of TGF-β signaling, we conclude that Smad4 is rapidly and continuously shuttling between the nucleus and the cytoplasm, the distribution of Smad4 between the nucleus and the cytoplasm being dictated by the relative strengths of the nuclear import and export signals. We demonstrate that inhibition of CRM1-mediated nuclear export by treatment of cells with leptomycin B results in endogenous Smad4 accumulating very rapidly in the nucleus. Endogenous Smad2 and Smad3 are completely unaffected by leptomycin B treatment, indicating that the nucleocytoplasmic shuttling is specific for Smad4. We propose that, upon TGF-β signaling, complex formation between Smad4 and activated Smad2 or -3 leads to nuclear accumulation of Smad4 through inhibition of its nuclear export. We demonstrate that after prolonged TGF-β signaling Smad2 becomes dephosphorylated and Smad2 and Smad4 accumulate back in the cytoplasm

Loss of Smad4 function in pancreatic tumors: C-terminal truncation leads to decreased stability.

At early stages of tumorigenesis, the transforming growth factor-beta (TGF-beta) signaling pathway is thought to have tumor suppressor activity as a result of its ability to arrest the growth of epithelial cells. Smad4 plays a pivotal role in the TGF-beta signaling pathway and has been identified as a tumor suppressor, being mutated or deleted in approximately 50% of pancreatic carcinomas and 15% of colorectal cancers. A nonsense mutation generating a C-terminal truncation of 38 amino acids in the Smad4 protein has been identified in a pancreatic adenocarcinoma (Hahn, S. A., Schutte, M., Hoque, A. T., Moskaluk, C. A., da Costa, L. T., Rozenblum, E., Weinstein, C. L., Fischer, A., Yeo, C. J., Hruban, R. H., and Kern, S. E. (1996) Science 271, 350-353), and here we investigate the functional consequences of this mutation. We demonstrate that the C-terminal truncation prevents Smad4 homomeric complex formation and heteromeric complex formation with activated Smad2. Furthermore, the mutant protein is unable to be recruited to DNA by transcription factors and hence cannot form transcriptionally active DNA-binding complexes. These observations are supported by molecular modeling, which indicates that the truncation removes residues critical for homomeric and heteromeric Smad complex formation. We go on to show that the mutant Smad4 is highly unstable compared with wild type Smad4 and is rapidly degraded through the ubiquitin-proteasome pathway. Consistent with this, we demonstrate that the pancreatic adenocarcinoma harboring this mutated allele, in conjunction with loss of the other allele, expresses no Smad4 protein. Thus we conclude that these tumors completely lack Smad4 activity

Effects of estradiol, progesterone or cAMP on expression of PGRMC1 and progesterone receptor in a xenograft model of human endometrium and in endometrial cell culture

Estradiol and progesterone are key regulators of the menstrual cycle. In the human endometrium, progesterone induces morphological changes required for blastocyst implantation. Dysregulated response to progesterone can lead to endometrial pathologies including uterine bleeding and endometriosis. Besides the canonical nuclear progesterone receptor (encoded by the PGR gene), alternative response pathways include Progesterone Receptor Membrane Component 1 (PGRMC1), suspected to be involved in pathogenesis of endometrial diseases. We previously reported the spatiotemporal profile of PGRMC1 expression in the human endometrium along the menstrual cycle, highlighting progressive increase and decrease during the proliferative and secretory phases, respectively. Here we directly addressed its regulation by estradiol and progesterone, with systematic comparison with regulation of PGR expression. We found a direct correlation between expression of both genes during the proliferative and secretory phases in the cycling endometrium, but not during the menstrual phase. In a xenograft model mimicking the cycle phases, estradiol significantly increased and progesterone significantly decreased PGR expression but changes were not significant for PGRMC1. Finally, we did not find any significant effect of the ovarian steroids on expression of PGR or PGRMC1 in primary culture of endometrial stromal cells, except for a small increase in PGR expression by estradiol. Altogether, our experiments do not allow a major advance in our understanding of the mechanisms of cyclic variation of PGRMC1 expression, in particular regarding potential regulation by the ovarian steroids