Description of the senescent human dermal fibroblasts secretome and of their impact on early step of skin carcinogenesis

Bien que le risque de développer un carcinome augmente avec le vieillissement, la sénescence reste considérée comme un mécanisme suppresseur de tumeurs. Contrairement aux fibroblastes de derme, les kératinocytes primaires humains (NHEK) ne sont pas bloqués irréversiblement au plateau de sénescence in vitro. Une fraction de NHEK sénescents échappe à la mort et génère de nouvelles cellules néoplasiques. Le développement des cancers peut être stimulé par leur micro-environnement vieillissant pro-inflammatoire produit principalement par les fibroblastes sénescents. Mon projet de thèse avait pour but de caractériser le sécrétome des fibroblastes sénescents de derme et d’en étudier l’impact sur l’émergence néoplasique des NHEK. Nous avons montré que les fibroblastes sénescents de derme produisent un sécrétome pro-inflammatoire : sécrétion de cytokines (MCP-1 ; IL-6), de facteurs de croissance (HGF/SF) et de métalloprotéases (MMP-1 ; -2…). Le conditionnement des cultures des NHEK par ce sécrétome des fibroblastes sénescents stimule la transition épithélium-mésenchyme et la migration des NHEK néoplasiques. Les MMP-1 et -2, sécrétées par les fibroblastes sénescents sont à l’origine des capacités migratoires de ces NHEK néoplasiques en activant leur récepteur à la thrombine PAR-1 (Proteases Activated Receptor), généralement associé aux stades tardifs. La caractérisation du sécrétome des fibroblastes sénescents par une analyse protéomique, montre aussi un défaut de sécrétion de plusieurs glycoprotéines de la matrice extracellulaire, aux propriétés anti-tumorales, provoqué par l’activation des voies de réponse au stress du réticulum endoplasmique associé à la sénescence.While the risk to develop a carcinoma increased with aging, cellular senescence is consider as a potent barrier to tumorigenesis. Contrarily to the fibroblasts, senescence of normal human primary epidermal keratinocytes (NHEK) is not irreversible. Indeed, a fraction of NHEK spontaneously escapes from the senescence plateau and grows as neoplastic cells. Carcinoma development could be enhanced by the aging pro-inflammatory micro-environment produced principally by senescent fibroblasts. The aim of my thesis was to describe the characteristics of the secretome of senescent dermal human fibroblasts (NHDF) and then to determine whether it could enhanced early neoplastic step of keratinocytes. We showed that senescent NHDF produce a pro-inflammatory secretome with secretion of cytokines, growth factors and metalloproteinases (MMPs). Conditioning of keratinocytes by senescent NHDF secretome enhanced the early transformation of the neoplastic keratinocytes by promoting Epithelial-to-Mesenchymal Transition and migration ability. Particularly, MMP-1; -2 specifically secreted by senescent NHDF promote the early migratory capacity of these NHEK, by activating the protease-activated receptor-1 (thrombin receptor, PAR-1), up to now associated to more aggressive cancer. The characterization of senescent NHDF conditioned media by a proteomic analysis highlighted a loss of secretion of several extracellular matrix glycoproteins which have some important anti-tumoral proprieties. Finally, we demonstrated that the defective secretions of these glycoproteins by senescent NHDF are the result of a constitutive endoplasmic reticulum stress and activation of the Unfolded Response Pathway

Caractérisation du sécrétome des fibroblastes sénescents de derme humains et de son impact sur les étapes initiales de la carcinogenèse de la peau

Bien que le risque de développer un carcinome augmente avec le vieillissement, la sénescence reste considérée comme un mécanisme suppresseur de tumeurs. Contrairement aux fibroblastes de derme, les kératinocytes primaires humains (NHEK) ne sont pas bloqués irréversiblement au plateau de sénescence in vitro. Une fraction de NHEK sénescents échappe à la mort et génère de nouvelles cellules néoplasiques. Le développement des cancers peut être stimulé par leur micro-environnement vieillissant pro-inflammatoire produit principalement par les fibroblastes sénescents. Mon projet de thèse avait pour but de caractériser le sécrétome des fibroblastes sénescents de derme et d en étudier l impact sur l émergence néoplasique des NHEK. Nous avons montré que les fibroblastes sénescents de derme produisent un sécrétome pro-inflammatoire : sécrétion de cytokines (MCP-1 ; IL-6), de facteurs de croissance (HGF/SF) et de métalloprotéases (MMP-1 ; -2 ). Le conditionnement des cultures des NHEK par ce sécrétome des fibroblastes sénescents stimule la transition épithélium-mésenchyme et la migration des NHEK néoplasiques. Les MMP-1 et -2, sécrétées par les fibroblastes sénescents sont à l origine des capacités migratoires de ces NHEK néoplasiques en activant leur récepteur à la thrombine PAR-1 (Proteases Activated Receptor), généralement associé aux stades tardifs. La caractérisation du sécrétome des fibroblastes sénescents par une analyse protéomique, montre aussi un défaut de sécrétion de plusieurs glycoprotéines de la matrice extracellulaire, aux propriétés anti-tumorales, provoqué par l activation des voies de réponse au stress du réticulum endoplasmique associé à la sénescence.While the risk to develop a carcinoma increased with aging, cellular senescence is consider as a potent barrier to tumorigenesis. Contrarily to the fibroblasts, senescence of normal human primary epidermal keratinocytes (NHEK) is not irreversible. Indeed, a fraction of NHEK spontaneously escapes from the senescence plateau and grows as neoplastic cells. Carcinoma development could be enhanced by the aging pro-inflammatory micro-environment produced principally by senescent fibroblasts. The aim of my thesis was to describe the characteristics of the secretome of senescent dermal human fibroblasts (NHDF) and then to determine whether it could enhanced early neoplastic step of keratinocytes. We showed that senescent NHDF produce a pro-inflammatory secretome with secretion of cytokines, growth factors and metalloproteinases (MMPs). Conditioning of keratinocytes by senescent NHDF secretome enhanced the early transformation of the neoplastic keratinocytes by promoting Epithelial-to-Mesenchymal Transition and migration ability. Particularly, MMP-1; -2 specifically secreted by senescent NHDF promote the early migratory capacity of these NHEK, by activating the protease-activated receptor-1 (thrombin receptor, PAR-1), up to now associated to more aggressive cancer. The characterization of senescent NHDF conditioned media by a proteomic analysis highlighted a loss of secretion of several extracellular matrix glycoproteins which have some important anti-tumoral proprieties. Finally, we demonstrated that the defective secretions of these glycoproteins by senescent NHDF are the result of a constitutive endoplasmic reticulum stress and activation of the Unfolded Response Pathway.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Selective handling of droplets in a microfluidic device using magnetic rails

International audienc

Senolytic Targeting of Bcl-2 Anti-Apoptotic Family Increases Cell Death in Irradiated Sarcoma Cells

Radiotherapy (RT) is a key component of cancer treatment. Most of the time, radiation is given after surgery but for soft-tissue sarcomas (STS), pre-surgical radiation is commonly utilized. However, despite improvements in RT accuracy, the rate of local recurrence remains high and is the major cause of death for patients with STS. A better understanding of cell fates in response to RT could provide new therapeutic options to enhance tumour cell killing by RT and facilitate surgical resection. Here, we showed that irradiated STS cell cultures do not die but instead undergo therapy-induced senescence (TIS), which is characterized by proliferation arrest, senescence-associated β-galactosidase activity, secretion of inflammatory cytokines and persistent DNA damage. STS-TIS was also associated with increased levels of the anti-apoptotic Bcl-2 family of proteins which rendered cells targetable using senolytic Bcl-2 inhibitors. As oppose to radiation alone, the addition of senolytic agents Venetoclax (ABT-199) or Navitoclax (ABT-263) after irradiation induced a rapid apoptotic cell death in STS monolayer cultures and in a more complex three-dimensional culture model. Together, these data suggest a new promising therapeutic approach for sarcoma patients who receive neoadjuvant RT. The addition of senolytic agents to radiation treatments may significantly reduce tumour volume prior to surgery and thereby improve the clinical outcome of patients

Senolytic Targeting of Bcl-2 Anti-Apoptotic Family Increases Cell Death in Irradiated Sarcoma Cells

Radiotherapy (RT) is a key component of cancer treatment. Most of the time, radiation is given after surgery but for soft-tissue sarcomas (STS), pre-surgical radiation is commonly utilized. However, despite improvements in RT accuracy, the rate of local recurrence remains high and is the major cause of death for patients with STS. A better understanding of cell fates in response to RT could provide new therapeutic options to enhance tumour cell killing by RT and facilitate surgical resection. Here, we showed that irradiated STS cell cultures do not die but instead undergo therapy-induced senescence (TIS), which is characterized by proliferation arrest, senescence-associated β-galactosidase activity, secretion of inflammatory cytokines and persistent DNA damage. STS-TIS was also associated with increased levels of the anti-apoptotic Bcl-2 family of proteins which rendered cells targetable using senolytic Bcl-2 inhibitors. As oppose to radiation alone, the addition of senolytic agents Venetoclax (ABT-199) or Navitoclax (ABT-263) after irradiation induced a rapid apoptotic cell death in STS monolayer cultures and in a more complex three-dimensional culture model. Together, these data suggest a new promising therapeutic approach for sarcoma patients who receive neoadjuvant RT. The addition of senolytic agents to radiation treatments may significantly reduce tumour volume prior to surgery and thereby improve the clinical outcome of patients

Non-canonical ATM/MRN activities temporally define the senescence secretory program.

Senescent cells display senescence-associated (SA) phenotypic programs such as stable proliferation arrest (SAPA) and a secretory phenotype (SASP). Senescence-inducing persistent DNA double-strand breaks (pDSBs) cause an immediate DNA damage response (DDR) and SAPA, but the SASP requires days to develop. Here, we show that following the immediate canonical DDR, a delayed chromatin accumulation of the ATM and MRN complexes coincides with the expression of SASP factors. Importantly, histone deacetylase inhibitors (HDACi) trigger SAPA and SASP in the absence of DNA damage. However, HDACi-induced SASP also requires ATM/MRN activities and causes their accumulation on chromatin, revealing a DNA damage-independent, non-canonical DDR activity that underlies SASP maturation. This non-canonical DDR is required for the recruitment of the transcription factor NF-κB on chromatin but not for its nuclear translocation. Non-canonical DDR further does not require ATM kinase activity, suggesting structural ATM functions. We propose that delayed chromatin recruitment of SASP modulators is the result of non-canonical DDR signaling that ensures SASP activation only in the context of senescence and not in response to transient DNA damage-induced proliferation arrest

Senescent Fibroblasts Enhance Early Skin Carcinogenic Events via a Paracrine MMP-PAR-1 Axis

<div><p>The incidence of carcinoma increases greatly with aging, but the cellular and molecular mechanisms underlying this correlation are only partly known. It is established that senescent fibroblasts promote the malignant progression of already-transformed cells through secretion of inflammatory mediators. We investigated here whether the senescent fibroblast secretome might have an impact on the very first stages of carcinogenesis. We chose the cultured normal primary human epidermal keratinocyte model, because after these cells reach the senescence plateau, cells with transformed and tumorigenic properties systematically and spontaneously emerge from the plateau. In the presence of medium conditioned by autologous senescent dermal fibroblasts, a higher frequency of post-senescence emergence was observed and the post-senescence emergent cells showed enhanced migratory properties and a more marked epithelial-mesenchymal transition. Using pharmacological inhibitors, siRNAs, and blocking antibodies, we demonstrated that the MMP-1 and MMP-2 matrix metalloproteinases, known to participate in late stages of cancer invasion and metastasis, are responsible for this enhancement of early migratory capacity. We present evidence that MMPs act by activating the protease-activated receptor 1 (PAR-1), whose expression is specifically increased in post-senescence emergent keratinocytes. The physiopathological relevance of these results was tested by analyzing MMP activity and PAR-1 expression in skin sections. Both were higher in skin sections from aged subjects than in ones from young subjects. Altogether, our results suggest that during aging, the dermal and epidermal skin compartments might be activated coordinately for initiation of skin carcinoma, via a paracrine axis in which MMPs secreted by senescent fibroblasts promote very early epithelial-mesenchymal transition of keratinocytes undergoing transformation and oversynthesizing the MMP-activatable receptor PAR-1.</p></div