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Julien Sage

Publications and source records attributed to Julien Sage.

16 recordsLinked to original sources
PubMed2025

Functional synapses between neurons and small cell lung cancer.

Small cell lung cancer (SCLC) is a highly aggressive type of lung cancer, characterized by rapid proliferation, early metastatic spread, frequent early relapse and a high mortality rate1-3. Recent evidence has suggested that innervation has an important role in the development and progression of several types of cancer4,5. Cancer-to-neuron synapses have been reported in gliomas6,7, but whether peripheral tumours can form such structures is unknown. Here we show that SCLC cells can form functional synapses and receive synaptic transmission. Using in vivo insertional mutagenesis screening in conjunction with cross-species genomic and transcriptomic validation, we identified neuronal, synaptic and glutamatergic signalling gene sets in mouse and human SCLC. Further experiments revealed the ability of SCLC cells to form synaptic structures with neurons in vitro and in vivo. Electrophysiology and optogenetic experiments confirmed that cancer cells can receive NMDA receptor- and GABAA receptor-mediated synaptic inputs. Fitting with a potential oncogenic role of neuron-SCLC interactions, we showed that SCLC cells derive a proliferation advantage when co-cultured with vagal sensory or cortical neurons. Moreover, inhibition of glutamate signalling had therapeutic efficacy in an autochthonous mouse model of SCLC. Therefore, following malignant transformation, SCLC cells seem to hijack synaptic signalling to promote tumour growth, thereby exposing a new route for therapeutic intervention.

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PubMed2025

Efficient and multiplexed somatic genome editing with Cas12a mice.

Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations. However, existing models have a limited ability to create multiple targeted edits, hindering our understanding of complex genetic interactions. Here we generate transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a), which robustly generates compound genotypes, including diverse cancers driven by inactivation of trios of tumour suppressor genes or an oncogenic translocation. We integrate these modular CRISPR RNA (crRNA) arrays with clonal barcoding to quantify the size and number of tumours with each array, as well as the impact of varying the guide number and position within a four-guide array. Finally, we generate tumours with inactivation of all combinations of nine tumour suppressor genes and find that the fitness of triple-knockout genotypes is largely explainable by one- and two-gene effects. These Cas12a alleles will enable further rapid creation of disease models and high-throughput investigation of coincident genomic alterations in vivo.

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PubMed2007

pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16INK4alpha tumor suppressor gene.

Genetic studies have demonstrated that Bmi1 promotes cell proliferation and stem cell self-renewal with a correlative decrease of p16(INK4a) expression. Here, we demonstrate that Polycomb genes EZH2 and BMI1 repress p16 expression in human and mouse primary cells, but not in cells deficient for pRB protein function. The p16 locus is H3K27-methylated and bound by BMI1, RING2, and SUZ12. Inactivation of pRB family proteins abolishes H3K27 methylation and disrupts BMI1, RING2, and SUZ12 binding to the p16 locus. These results suggest a model in which pRB proteins recruit PRC2 to trimethylate p16, priming the BMI1-containing PRC1L ubiquitin ligase complex to silence p16.

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PubMed2005

The related retinoblastoma (pRb) and p130 proteins cooperate to regulate homeostasis in the intestinal epithelium.

pRb, p107, and p130 are related proteins that play a central role in the regulation of cell cycle progression and terminal differentiation in mammalian cells. Nevertheless, it is still largely unclear how these proteins achieve this regulation in vivo. The intestinal epithelium is an ideal in vivo system in which to study the molecular pathways that regulate proliferation and differentiation because it exists in a constant state of development throughout an animal's lifetime. We studied the phenotypic effects on the intestinal epithelium of mutating Rb and p107 or p130. Although mutating these genes singly had little or no effect, loss of pRb and p107 or p130 together produced chronic hyperplasia and dysplasia of the small intestinal and colonic epithelium. In Rb/p130 double mutants this hyperplasia was associated with defects in terminal differentiation of specific cell types and was dependent on the increased proliferation seen in the epithelium of mutant animals. At the molecular level, dysregulation of the Rb pathway led to an increase in the expression of Math1, Cdx1, Cdx2, transcription factors that regulate proliferation and differentiation in the intestinal epithelium. The absence of Cdx1 function in Rb/p130 double mutant mice partially reverted the histologic phenotype by suppressing ectopic mitosis in the epithelium. These studies implicate the Rb pathway as a regulator of epithelial homeostasis in the intestine.

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PubMed2005

C/EBPbeta cooperates with RB:E2F to implement Ras(V12)-induced cellular senescence.

In primary cells, overexpression of oncogenes such as Ras(V12) induces premature senescence rather than transformation. Senescence is an irreversible form of G1 arrest that requires the p19ARF/p53 and p16INK4a/pRB pathways and may suppress tumorigenesis in vivo. Here we show that the transcription factor C/EBPbeta is required for Ras(V12)-induced senescence. C/EBPbeta-/- mouse embryo fibroblasts (MEFs) expressing Ras(V12) continued to proliferate despite unimpaired induction of p19ARF and p53, and lacked morphological features of senescent fibroblasts. Enforced C/EBPbeta expression inhibited proliferation of wild-type MEFs and also slowed proliferation of p19Arf-/- and p53-/- cells, indicating that C/EBPbeta acts downstream or independently of p19ARF/p53 to suppress growth. C/EBPbeta was unable to inhibit proliferation of MEFs lacking all three RB family proteins or wild-type cells expressing dominant negative E2F-1 and, instead, stimulated their growth. C/EBPbeta decreased expression of several E2F target genes and was associated with their promoters in chromatin immunoprecipitation assays, suggesting that C/EBPbeta functions by repressing genes required for cell cycle progression. C/EBPbeta is therefore a novel component of the RB:E2F-dependent senescence program activated by oncogenic stress in primary cells.

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PubMed2005

Making young tumors old: a new weapon against cancer?

As the population of industrial nations ages, the incidence of cancer and cancer mortality is increasing. Intuitively, older organisms may be less able to cope with accumulated damage and thus be more prone to develop cancer. However, so far, the links between aging and cancer have been only partially explored. Strikingly, four recent studies now indicate that premature senescence accompanied by cell cycle arrest occurs in tumors initiated by an oncogenic mutation. Thus, senescence may act as a key tumor suppressor mechanism in young tumors in vivo.

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PubMed2004

Cell type-specific effects of Rb deletion in the murine retina.

Certain cells of the human retina are extremely sensitive to loss of function of the retinoblastoma tumor suppressor gene RB. Retinoblastomas develop early in life and at high frequency in individuals heterozygous for a germ-line RB mutation, and sporadic retinoblastomas invariably have somatic mutation in the RB gene. In contrast, retinoblastomas do not develop in Rb+/- mice. Although retinoblastoma is thought to have developmental origins, the function of Rb in retinal development has not been fully characterized. Here we studied the role of Rb in normal retinal development and in retinoblastoma using conditional Rb mutations in the mouse. In late embryogenesis, Rb-deficient retinas exhibited ectopic S-phase and high levels of p53-independent apoptosis, particularly in the differentiating retinal ganglion cell layer. During postnatal retinal development, loss of Rb led to more widespread retinal apoptosis, and adults showed loss of photoreceptors and bipolar cells. Conditional Rb mutation in the retina did not result in retinoblastoma formation even in a p53-mutant background. However, on a p107- or p130-deficient background, Rb mutation in the retina caused retinal dysplasia or retinoblastoma.

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PubMed2004

Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents.

The retinoblastoma (RB) tumor suppressor has been proposed to function as a key mediator of cell cycle checkpoints induced by chemotherapeutic agents. However, these prior studies have relied on embryonic fibroblasts harboring chronic loss of RB, a condition under which compensation of RB functions is known to occur. Here we utilized primary adult fibroblasts derived from mice harboring loxP sites flanking exon 3 of the Rb gene to delineate the action of RB in the chemotherapeutic response. In this system we find that targeted disruption of Rb leads to little overt change in cell cycle distribution. However, these cells exhibited deregulation of RB/E2F target genes and became aneuploid following culture in the absence of RB. When challenged with both DNA damaging and antimetabolite chemotherapeutics, RB was required for primary adult cells to undergo DNA damage checkpoint responses and loss of RB resulted in enhanced aneuploidy following challenge. In contrast, following spontaneous immortalization and the loss of functional p53 signaling, the antimetabolite 5-fluorouracil (5-FU) failed to induce arrest despite the presence of RB. In these immortal cultures RB/E2F targets were deregulated in a complex, gene-specific manner and RB was required for the checkpoint response to camptothecin (CPT). Mechanistic analyses of the checkpoint responses in primary cells indicated that loss of RB leads to increased p53 signaling and decreased viability following both CPT and 5-FU treatment. However, the mechanism through which these agents act to facilitate cell cycle inhibition through RB were distinct. These studies underscore the critical role of RB in DNA-damage checkpoint signaling and demonstrate that RB mediates chemotherapeutic-induced cell cycle inhibition in adult fibroblasts by distinct mechanisms.

Aneuploidy↗
PubMed2004

RB signaling prevents replication-dependent DNA double-strand breaks following genotoxic insult.

Cell cycle checkpoints induced by DNA damage play an integral role in preservation of genomic stability by allowing cells to limit the propagation of deleterious mutations. The retinoblastoma tumor suppressor (RB) is crucial for the maintenance of the DNA damage checkpoint function because it elicits cell cycle arrest in response to a variety of genotoxic stresses. Although sporadic loss of RB is characteristic of most cancers and results in the bypass of the DNA damage checkpoint, the consequence of RB loss upon chemotherapeutic responsiveness has been largely uninvestigated. Here, we employed a conditional knockout approach to ablate RB in adult fibroblasts. This system enabled us to examine the DNA damage response of adult cells following acute RB deletion. Using this system, we demonstrated that loss of RB disrupted the DNA damage checkpoint elicited by either cisplatin or camptothecin exposure. Strikingly, this bypass was not associated with enhanced repair, but rather the accumulation of phosphorylated H2AX (gammaH2AX) foci, which indicate DNA double-strand breaks. The formation of gammaH2AX foci was due to ongoing replication following chemotherapeutic treatment in the RB-deficient cells. Additionally, peak gammaH2AX accumulation occurred in S-phase cells undergoing DNA replication in the presence of damage, and these gammaH2AX foci co-localized with replication foci. These results demonstrate that acute RB loss abrogates DNA damage-induced cell cycle arrest to induce gammaH2AX foci formation. Thus, secondary genetic lesions induced by RB loss have implications for the chemotherapeutic response and the development of genetic instability.

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PubMed2004

Cyclin C makes an entry into the cell cycle.

From yeast to humans, cell cycle progression is orchestrated by the oscillation of kinase activities associated with cyclins. In an article published recently in Cell, Ren and Rollins investigate mechanisms controlling the G0/G1 transition in quiescent cells and identify new cyclin C/Cdk3 complexes as key regulators of cell cycle reentry in human cells.

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PubMed2003

Perp is a mediator of p53-dependent apoptosis in diverse cell types.

The induction of apoptosis by the p53 protein is critical for its activity as a tumor suppressor. Although it is clear that p53 induces apoptosis at least in part by direct transcriptional activation of target genes, the set of p53 target genes that mediate p53 function in apoptosis in vivo remains to be well defined. The Perp (p53 apoptosis effector related to PMP-22) gene is highly expressed in cells undergoing p53-dependent apoptosis as compared to cells undergoing p53-dependent G1 arrest. Perp is a direct p53 target, and its overexpression is sufficient to induce cell death in fibroblasts, implicating it as an important component of p53 apoptotic function. Here, through the generation of Perp-deficient mice, we analyze the role of Perp in the p53 apoptosis pathway in multiple primary cell types by comparing the cell death responses of Perp null cells to those of wild-type and p53 null cells. These experiments demonstrate the involvement of Perp in p53-mediated cell death in thymocytes and neurons but not in E1A-expressing MEFs, indicating a cell type-specific role for Perp in the p53 cell death pathway. In addition, we show that Perp is not required for proliferation-associated functions of p53. Thus, Perp selectively mediates the p53 apoptotic response, and the requirement for Perp is dictated by cellular context.

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PubMed2003

Acute mutation of retinoblastoma gene function is sufficient for cell cycle re-entry.

Cancer cells arise from normal cells through the acquisition of a series of mutations in oncogenes and tumour suppressor genes. Mouse models of human cancer often rely on germline alterations that activate or inactivate genes of interest. One limitation of this approach is that germline mutations might have effects other than somatic mutations, owing to developmental compensation. To model sporadic cancers associated with inactivation of the retinoblastoma (RB) tumour suppressor gene in humans, we have produced a conditional allele of the mouse Rb gene. We show here that acute loss of Rb in primary quiescent cells is sufficient for cell cycle entry and has phenotypic consequences different from germline loss of Rb function. This difference is explained in part by functional compensation by the Rb-related gene p107. We also show that acute loss of Rb in senescent cells leads to reversal of the cellular senescence programme. Thus, the use of conditional knockout strategies might refine our understanding of gene function and help to model human cancer more accurately.

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PubMed2003

Recapitulation of the effects of the human papillomavirus type 16 E7 oncogene on mouse epithelium by somatic Rb deletion and detection of pRb-independent effects of E7 in vivo.

Although the human papillomavirus (HPV) E7 oncogene is known to contribute to the development of human cervical cancer, the mechanisms of its carcinogenesis are poorly understood. The first identified and most recognized function of E7 is its binding to and inactivation of the retinoblastoma tumor suppressor (pRb), but at least 18 other biological activities have also been reported for E7. Thus, it remains unclear which of these many activities contribute to the oncogenic potential of E7. We used a Cre-lox system to abolish pRb expression in the epidermis of transgenic mice and compared the outcome with the effects of E7 expression in the same tissue at early ages. Mice lacking pRb in epidermis showed epithelial hyperplasia, aberrant DNA synthesis, and improper differentiation. In addition, Rb-deleted epidermis (i.e., epidermis composed of cells with Rb deleted) exhibited centrosomal abnormalities and failed to arrest the cell cycle in response to ionizing radiation. Transgenic mice expressing E7 in skin display the same range of phenotypes. In sum, few differences were detected between Rb-deleted epidermis and E7-expressing epidermis in young mice. However, when both E7 was expressed and Rb was deleted in the same tissue, increased hyperplasia and dysplasia were observed. These findings indicate that inactivation of the Rb pathway can largely account for E7's phenotypes at an early age, but that pRb-independent activities of E7 are detectable in vivo.

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PubMed2002

An induced Ets repressor complex regulates growth arrest during terminal macrophage differentiation.

Defining the molecular mechanisms that coordinately regulate proliferation and differentiation is a central issue in development. Here, we describe a mechanism in which induction of the Ets repressor METS/PE1 links terminal differentiation to cell cycle arrest. Using macrophages as a model, we provide evidence that METS/PE1 blocks Ras-dependent proliferation without inhibiting Ras-dependent expression of cell type-specific genes by selectively replacing Ets activators on the promoters of cell cycle control genes. Antiproliferative effects of METS require its interaction with DP103, a DEAD box-containing protein that assembles a novel corepressor complex. Functional interactions between the METS/DP103 complex and E2F/ pRB family proteins are also necessary for inhibition of cellular proliferation, suggesting a combinatorial code that directs permanent cell cycle exit during terminal differentiation.

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PubMed2002

Targeted point mutations of p53 lead to dominant-negative inhibition of wild-type p53 function.

The p53 tumor suppressor gene is the most frequently mutated gene in human cancers, and germ-line p53 mutations cause a familial predisposition for cancer. Germ-line or sporadic p53 mutations are usually missense and typically affect the central DNA-binding domain of the protein. Because p53 functions as a tetrameric transcription factor, mutant p53 is thought to inhibit the function of wild-type p53 protein. Here, we studied the possible dominant-negative inhibition of wild-type p53 protein by two different, frequently occurring point mutations. The R270H and P275S mutations were targeted into the genome of mouse embryonic stem cells to allow the analysis of the effects of the mutant proteins expressed in normal cells at single-copy levels. In embryonic stem cells, the presence of a heterozygous point-mutated allele resulted in delayed transcriptional activation of several p53 downstream target genes on exposure to gamma irradiation. Doxorubicin-induced apoptosis was severely affected in the mutant embryonic stem cells compared with wild-type cells. Heterozygous mutant thymocytes had a severe defect in p53-dependent apoptotic pathways after treatment with gamma irradiation or doxorubicin, whereas p53-independent apoptotic pathways were intact. Together these data demonstrate that physiological expression of point-mutated p53 can strongly limit overall cellular p53 function, supporting the dominant-negative action of such mutants. Also, cells heterozygous for such mutations may be compromised in terms of tumor suppression and response to chemotherapeutic agents.

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