Procathepsin L secretion, which triggers tumor progression, is regulated by Rab4A in human melanoma cells

International audienceThe switch of human melanoma cell phenotype from non to highly tumorigenic and metastatic is triggered by the increase of procathepsin L secretion which modifies tumor microenvironment. Our aim was to identify components involved in the regulation of procathepsin L secretion in melanoma cells. We focused on Rab family members, i.e. Rab3A, Rab4A, Rab4B, Rab5A, Rab8A, Rab11A, Rab27A and Rab33A, involved in distinct regulatory pathways. From analysis of mRNA and protein expression of these Rab components and their knock down by specific siRNAs emerged that Rab4A protein is involved in the regulation of procathepsin L secretion. This result was strengthened as procathepsin L secretion was either inhibited by expression of Rab4A dominant negative mutant or increased by overexpression of the Rab4A wild type. Rab4A regulation: 1) discriminates between procathepsin L secretion and expression of intracellular cathepsin L forms; 2) did not modify other Rab proteins and GAPDH expression or IL-8 and MMP2 secretion; 3) still efficient on unglycosylated procathepsin L secretion. Thus, down- or up-regulation of Rab4A expression or Rab4A function triggered inhibition or increase of procathepsin L secretion, respectively. Furthermore, Rab4A regulation, by modifying procathepsin L secretion, switches the tumorigenic phenotype of human melanoma cells in nude mice

Contribution à l'identification des mécanismes impliqués dans la sécrétion de la procathepsine L, dont l'augmentation stimule le pouvoir tumorigène et métastique des mélanomes humains

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF

Les fonctions innées des lymphocytes T CD8 dans la lutte contre le cancer

International audienceno abstrac

Allotransplantation Is Associated With Exacerbation of CD8 T-Cell Senescence: The Particular Place of the Innate CD8 T-Cell Component

International audienceImmunosenescence is a physiological process that is associated with changes in the immune system, particularly among CD8 T-cells. Recent studies have hypothesized that senescent CD8 T-cells are produced with chronologic age by chronic stimulation, leading to the acquisition of hallmarks of innate-like T-cells. While conventional CD8 T-cells are quite well characterized, CD8 T-cells sharing features of NK cells and memory CD8 T-cells, are a newly described immune cell population. They can be distinguished from conventional CD8 T-cells by their combined expression of panKIR/NKG2A and Eomesodermin (E), a unique phenotype closely associated with IFN-γ production in response to innate stimulation. Here, we first provided new evidence in favor of the innate character of panKIR/NKG2A(+) E(+) CD8 T-cells in normal subjects, documenting their position at an intermediate level in the innateness gradient in terms of both innate IFN-γ production and diminished mitochondrial mass. We also revealed that CD8 E(+) panKIR/NKG2A(+) T-cells, hereafter referred to as Innate E(+) CD8 T-cells, exhibit increased senescent (CD27(-) CD28(-)) phenotype, compared to their conventional memory counterparts. Surprisingly, this phenomenon was not dependent on age. Given that inflammation related to chronic viral infection is known to induce NK-like marker expression and a senescence phenotype among CD8 T-cells, we hypothesized that innate E(+) CD8 T-cells will be preferentially associated with exacerbated cellular senescence in response to chronic alloantigen exposure or CMV infection. Accordingly, in a pilot cohort of stable kidney allotransplant recipients, we observed an increased frequency of the Innate E(+) CD8 T-cell subset, together with an exacerbated senescent phenotype. Importantly, this phenotype cannot be explained by age alone, in clear contrast to their conventional memory counterparts. The senescent phenotype in CD8 T-cells was further increased in cytomegalovirus (CMV) positive serology transplant recipients, suggesting that transplantation and CMV, rather than aging by itself, may promote an exacerbated senescent phenotype of innate CD8 T-cells. In conclusion, we proposed that kidney transplantation, via the setting of inflammatory stimuli of alloantigen exposure and CMV infection, may exogenously age the CD8 T-cell compartment, especially its innate component. The physiopathological consequences of this change in the immune system remain to be elucidated

Human Papillomavirus 16 Oncoprotein E7 Stimulates UBF1-Mediated rDNA Gene Transcription, Inhibiting a p53-Independent Activity of p14^ARF

<div><p>High-risk human papillomavirus oncoproteins E6 and E7 play a major role in HPV-related cancers. One of the main functions of E7 is the degradation of pRb, while E6 promotes the degradation of p53, inactivating the p14^ARF-p53 pathway. pRb and p14^ARF can repress ribosomal DNA (rDNA) transcription in part by targeting the Upstream Binding Factor 1 (UBF1), a key factor in the activation of RNA polymerase I machinery. We showed, through ectopic expression and siRNA silencing of p14^ARF and/or E7, that E7 stimulates UBF1-mediated rDNA gene transcription, partly because of increased levels of phosphorylated UBF1, preventing the inhibitory function of p14^ARF. Unexpectedly, activation of rDNA gene transcription was higher in cells co-expressing p14^ARF and E7, compared to cells expressing E7 alone. We did not find a difference in P-UBF1 levels that could explain this data. However, p14^ARF expression induced E7 to accumulate into the nucleolus, where rDNA transcription takes place, providing an opportunity for E7 to interact with nucleolar proteins involved in this process. GST-pull down and co-immunoprecipitation assays showed interactions between p14^ARF, UBF1 and E7, although p14^ARF and E7 are not able to directly interact. Co-expression of a pRb-binding-deficient mutant (E7C24G) and p14^ARF resulted in EC24G nucleolar accumulation, but not in a significant higher activation of rDNA transcription, suggesting that the inactivation of pRb is involved in this phenomenon. Thus, p14^ARF fails to prevent E7-mediated UBF1 phosphorylation, but could facilitate nucleolar pRb inactivation by targeting E7 to the nucleolus. While others have reported that p19^ARF, the mouse homologue of p14^ARF, inhibits some functions of E7, we showed that E7 inhibits a p53-independent function of p14^ARF. These results point to a mutually functional interaction between p14^ARF and E7 that might partly explain why the sustained p14^ARF expression observed in most cervical pre-malignant lesions and malignancies may be ineffective.</p></div

Innate T-αβ lymphocytes as new immunological components of anti-tumoral "off-target" effects of the tyrosine kinase inhibitor dasatinib

International audienceKinase inhibitors hold great potential as targeted therapy against malignant cells. Among them, the tyrosine kinase inhibitor dasatinib is known for a number of clinically relevant off-target actions, attributed in part to effects on components of the immune system, especially conventional T-cells and natural killer (NK)-cells. Here, we have hypothesized that dasatinib also influences non-conventional T-αβ cell subsets known for their potential anti-tumoral properties, namely iNKT cells and the distinct new innate CD8 T-cell subset. In mice, where the two subsets were originally characterized, an activated state of iNKT cells associated with a shift toward an iNKT cell Th1-phenotype was observed after dasatinib treatment in vivo. Despite decreased frequency of the total memory CD8 T-cell compartment, the proportion of innate-memory CD8 T-cells and their IFNγ expression in response to an innate-like stimulation increased in response to dasatinib. Lastly, in patients administered with dasatinib for the treatment of BCR-ABL-positive leukemias, we provided the proof of concept that the kinase inhibitor also influences the two innate T-cell subsets in humans, as attested by their increased frequency in the peripheral blood. These data highlight the potential immunostimulatory capacity of dasatinib on innate T-αβ cells, thereby opening new opportunities for chemoimmunotherapy

Interleukin-1 Family Cytokines: Keystones in Liver Inflammatory Diseases

International audienceThe pyrogenic property being the first activity described, members of the interleukin-1 superfamily (IL-1α, IL-1β, IL-18, and the newest members: IL-33, IL-36, IL-37, and IL-38) are now known to be involved in several inflammatory diseases such as obesity, atherosclerosis, cancer, viral and parasite infections, and auto-inflammatory syndromes as well as liver diseases. Inflammation processes are keystones of chronic liver diseases, of which the etiology may be viral or toxic, as in alcoholic or non-alcoholic liver diseases. Inflammation is also at stake in acute liver failure involving massive necrosis, and in ischemia-reperfusion injury in the setting of liver transplantation. The role of the IL-1 superfamily of cytokines and receptors in liver diseases can be either protective or pro-inflammatory, depending on timing and the environment. Our review provides an overview of current understanding of the IL-1 family members in liver inflammation, highlighting recent key investigations, and therapeutic perspectives. We have tried to apply the concept of trained immunity to liver diseases, based on the role of the members of the IL-1 superfamily, first of all IL-1β but also IL-18 and IL-33, in modulating innate lymphoid immunity carried by natural killer cells, innate lymphoid cells or innate T-αβ lymphocytes

Expression of HPV16 E6/E7 in human cervical keratinocytes induces p14^ARF expression early after transduction and enhances UBF1 phosphorylation.

<p>Human keratinocytes were transduced with retroviral vectors encoding E6 and E7 (LXSNE6E7) or with an empty vector (LXSN), and were then cultured with or without G418. Protein extracts were analysed by Western blot at various times, using antibodies against p14^ARF, UBF, p-UBF(Ser 388), actin and pRb.</p

rDNA promoter activity and level of UBF phosphorylation upon E7 and/or p14^ARF depletion in the HPV16 positive CaSki cervical carcinoma line.

<p>E7 and/or p14^ARF were depleted in the HPV16 CaSki cervical carcinoma cell line using siRNA and Lipofectamine 2000 reagent. CaSki cells were transiently transfected with the pHrDNA-IRES-Luc plasmid along with the <i>Renilla</i> internal control pRLTK, siRNA control (Si CTR), E7 siRNA and/or p14^ARF siRNA and lysed at 48 h. (A) Western blot analysis of CaSki cells extracts probed with antibodies to phosphorylated UBF (P-UBF Ser 388), p14^ARF and actin (as a loading control). Quantification of western blots was done by measuring the relative intensity of the bands compared to internal controls (Actin), the values given are in arbitrary units. (B) Expression of E7 mRNA assessed by quantitative real time RT-PCR. (C) rDNA promoter activity. Reporter activity was calculated as the ratio of firefly luciferase activity to reference <i>Renilla</i> luciferase activity, and normalized so that that luciferase activity in control vector transfected cells equaled 1. The data are representative of three independent experiments and are the means of triplicate samples +/− SD (**p<0.01).</p

HPV16 E7 expression stimulates UBF1 phosphorylation.

<p>(A), Western blot analysis of H358 WT cells transiently transfected with the pcDNA3.1 (Control), pJ4Ω16E7 (E7) or pcDNA3.1-p14ARF (p14^ARF) expression vector, or both and probed with antibodies to UBF, phosphorylated UBF (P-UBF Ser 388), p14^ARF and actin (as a loading control). (B), Western blot analysis of H358 Cl19 cells cultured with (+Dox) or without (-Dox) 1 mg/ml doxycyclin, transfected with pJ4Ω16E7 (E7), and probed with the indicated antibodies. (C), Western blot analysis of MCF7 cells transduced with LXSN, LXSNE7 (E7), LXSNE6 (E6), and LXSNE6E7 (E6+E7), selected with G418, and probed with the indicated antibodies. Quantification of western blots was done by measuring the relative intensity of the bands compared to internal controls (Actin), the values given are in arbitrary units. Expression of E7 was monitored by quantitative real time RT-PCR. Western blot and qRT-PCR are representative of three experiments at least.</p