β-Catenin-Independent Activation of TCF1/LEF1 in Human Hematopoietic Tumor Cells through Interaction with ATF2 Transcription Factors

The role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of β-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to β-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of β-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of β-catenin. By a complementary approach in vivo, TCF1 mutants, which lacked the ability to bind to β-catenin, induced Xenopus embryo axis duplication, a hallmark of Wnt activation, and the expression of the Wnt target gene Xnr3. Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the β-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of β-catenin stabilization, thus establishing a new mechanism for TCF1/LEF1 transcriptional activity distinct from that associated with canonical Wnt signaling

Canonical Wnts function as potent regulators of osteogenesis by human mesenchymal stem cells

Genetic evidence indicates that Wnt signaling is critically involved in bone homeostasis. In this study, we investigated the functions of canonical Wnts on differentiation of adult multipotent human mesenchymal stem cells (hMSCs) in vitro and in vivo. We observe differential sensitivities of hMSCs to Wnt inhibition of osteogenesis versus adipogenesis, which favors osteoblastic commitment under binary in vitro differentiation conditions. Wnt inhibition of osteogenesis is associated with decreased expression of osteoblastic transcription factors and inhibition of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation, which are involved in osteogenic differentiation. An hMSC subpopulation exhibits high endogenous Wnt signaling, the inhibition of which enhances osteogenic and adipogenic differentiation in vitro. In an in vivo bone formation model, high levels of Wnt signaling inhibit de novo bone formation by hMSCs. However, hMSCs with exogenous expression of Wnt1 but not stabilized β-catenin markedly stimulate bone formation by naive hMSCs, arguing for an important role of a canonical Wnt gradient in hMSC osteogenesis in vivo

CRISPR/Cas9 editing of the genome for cancer modeling

International audienceThe CRISPR/Cas9 revolution has democratized access to genome editing in many biological fields, including cancer research. Cancer results from the multistep accumulation of mutations that confer to the transformed cells certain biological hallmarks typical of the malignant phenotype. One of the major goals in cancer research is to characterize such mutations and assess their implication in the oncogenic process. Through CRISPR/Cas9 technology, genetic aberrations identified in a patient's tumor can now be easily recreated in experimental models, which can then be used for basic research or for more translational applications. Here we review the different CRISPR/Cas9 strategies that have been implemented to reca-pitulate oncogenic mutations in both in vitro and in vivo systems, including novel strategies to model tumor evolution and genetic heterogeneity

Etude des mécanismes moléculaires impliqués dans la différentiation des cellules sympatho-surrénaliennes (Effet du neuropeptide PACAP sur le transcriptome des cellules de phéochromocytome)

Le neuropeptide PACAP participe au contrôle de la prolifération et de la différenciation des neuroblastes sympathiques. Afin d'étudier le rôle de ce neuropeptide dans le développement des cellules sympatho-surrénaliennes, nous avons caractérisé le phénotype des cellules PC12 de phéochromocytome de rat après différenciation par le PACAP. Nous avons montré que le neuropeptide modifie les propriétés électriques, contrôle l'expression de gènes marqueurs neuronaux et neuroendocrines, et module l'activité de facteurs de transcription dans les cellules PC12. L'analyse du transcriptome des cellules PC12 différenciées par le PACAP a révélé que le neuropeptide régule l'expression de nombreux gènes, dont la plupart sont associés à la prolifération, la survie et l'adhésion cellulaires. Nous avons ensuite montré, pour la première fois, que l'un de ces gènes, dont la fonction était jusqu'alors inconnue, est impliqué dans un processus crucial au cours de la différenciation cellulaire. Grâce à cette étude, nous avons identifié plus d'une centaine de gènes cibles du PACAP dans les cellules PC12, dont la caractérisation permettra de mieux comprendre le rôle de ce neuropeptide dans la différenciation des cellules sympatho-surrénaliennes saines et tumorales.ROUEN-BU Sciences (764512102) / SudocSudocFranceF

Mechanisms of Acquired Resistance and Tolerance to EGFR Targeted Therapy in Non-Small Cell Lung Cancer

Non-small cell lung cancers (NSCLC) harboring activating mutations of the epidermal growth factor receptor (EGFR) are treated with specific tyrosine kinase inhibitors (EGFR-TKIs) of this receptor, resulting in clinically responses that can generally last several months. Unfortunately, EGFR-targeted therapy also favors the emergence of drug tolerant or resistant cells, ultimately resulting in tumor relapse. Recently, cellular barcoding strategies have arisen as a powerful tool to investigate the clonal evolution of these subpopulations in response to anti-cancer drugs. In this review, we provide an overview of the currently available treatment options for NSCLC, focusing on EGFR targeted therapy, and discuss the common mechanisms of resistance to EGFR-TKIs. We also review the characteristics of drug-tolerant persister (DTP) cells and the mechanistic basis of drug tolerance in EGFR-mutant NSCLC. Lastly, we address how cellular barcoding can be applied to investigate the response and the behavior of DTP cells upon EGFR-TKI treatment

Modeling intratumor heterogeneity through CRISPR-barcodes

International audienceWe have devised a barcoding strategy to recapitulate cancer evolution through the emergence of subclonal mutations of interest, whose effects can be monitored in a dynamic manner. This approach can be easily adapted for a variety of applications, including combined modeling of multiple mechanisms of drug resistance or repair of oncogenic driver mutations in addicted cancer cells

Chromatin Modifications Sequentially Enhance ErbB2 Expression in ErbB2-Positive Breast Cancers

ErbB2 gene amplification occurs in 20%–25% of breast cancers, and its therapeutic targeting has markedly improved survival of patients with breast cancer in the adjuvant setting. However, resistance to these therapies can develop. Because epigenetic mechanisms can importantly influence oncogene expression and be druggable as well, we investigated histone modifications that influence ErbB2 overexpression, independent of gene amplification. We demonstrate here that ErbB2-overexpressing breast carcinomas acquire the H3K4me3 mark on the erbB2 promoter and that receptor-amplified tumors further acquire the H3K9ac mark, which is dependent on H3K4me3 mark acquisition. Targeting WD repeat domain 5 (Wdr5), which is absolutely required for H3K4me3 enrichment, decreased ErbB2 overexpression, associated with a decrease in the H3K4me3 mark on the erbB2 promoter. Of note, Wdr5 silencing cooperated with trastuzumab or chemotherapy in specifically inhibiting the growth of ErbB2-positive breast tumor cells. Thus, our studies illuminate epigenetic steps in the selection for ErbB2 activation

Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) Promotes Both Survival and Neuritogenesis in PC12 Cells through Activation of Nuclear Factor κB (NF-κB) Pathway

International audienceThe pituitary adenylate cyclase-activating polypeptide (PACAP) is a trophic factor that promotes neuronal survival and neurite outgrowth. However, the signaling pathways and the transcriptional mechanisms involved are not completely elucidated. Our previous studies aimed at characterizing the transcriptome of PACAP-differentiated PC12 cells revealed an increase in the expression of nuclear factor κB2 (NF-κB2) gene coding for p100/p52 subunit of NF-κB transcription factor. Here, we examined the role of the NF-κB pathway in neuronal differentiation promoted by PACAP. We first showed that PACAP-driven survival and neuritic extension in PC12 cells are inhibited following NF-κB pathway blockade. PACAP stimulated both c-Rel and p52 NF-κB subunit gene expression and nuclear translocation, whereas c-Rel down-regulation inhibited cell survival and neuritogenesis elicited by the neuropeptide. PACAP-induced c-Rel nuclear translocation was inhibited by ERK1/2 and Ca2+ blockers. Furthermore, the neuropeptide stimulated NF-κB p100 subunit processing into p52, indicative of activation of the NF-κB alternative pathway. Taken together, our data show that PACAP promotes both survival and neuritogenesis in PC12 cells by activating NF-κB pathway, most likely via classical and alternative signaling cascades involving ERK1/2 kinases, Ca2+, and c-Rel/p52 dimers

Pituitary Adenylate Cyclase-Activating Polypeptide Stimulates Secretoneurin Release and Secretogranin II Gene Transcription in Bovine Adrenochromaffin Cells through Multiple Signaling Pathways and Increased Binding of Pre-Existing Activator Protein-1-Like Transcription Factors

International audienceSecretoneurin (SN) is a novel bioactive peptide that derives from the neuroendocrine protein secretogranin II (SgII) by pro-teolytic processing and participates in neuro-immune communication. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP-38) dose-dependently stimulates (EC 50 ϳ 3 nM) SN release (up to 4-fold) and SgII gene expression (up to 60-fold) in cultured bovine adrenochromaffin cells. The effect of PACAP on both SN secretion and SgII mRNA levels is rapid and long lasting. We analyzed in this neuroen-docrine cell model the transduction pathways involved in both SN secretion and SgII gene transcription in response to PACAP. The cytosolic calcium chelator BAPTA-AM and the nonselective calcium channel antagonist NiCl 2 equally inhibited both secretion of the peptide and transcription of the SgII gene, indicating a major contribution of calcium influx in PACAP-induced SN biosynthesis and release in chromaffin cells. Inhibition of protein kinase A (PKA) or C (PKC) also reduced PACAP-evoked SN release but did not alter the stimulatory effect of PACAP on SgII mRNA levels. Conversely, application of mitogen-activated protein kinase inhibitors suppressed PACAP-induced SgII gene expression. The effect of PACAP on SgII mRNA levels, like the effect of the PKC stimulator 12-O-tetradecanoylphorbol-13-acetate (TPA), was not affected by cycloheximide, whereas the effects of the PKA stimulator for-skolin or cell-depolarization by high K ϩ were significantly reduced by the protein synthesis inhibitor. PACAP and TPA both increased the binding activity of the SgII cAMP response element to transacting factors present in chromaffin cell nuclear extracts, which are recognized by antibodies to activator protein -1-related proteins. These data indicate that SN biosynthe-sis is regulated by PACAP in chromaffin cells through complex signaling cascades, suggesting that SN may play a function during trans-synaptic stimulation of the adrenal medulla