Le rôle de la protéine kinase dépendante de l’ADN (DNA-PK) dans le processus métastatique

La protéine kinase dépendante de l’ADN (DNA-PK) est une sérine-thréonine kinase qui est un élément essentiel dans la voie de réparation de l’ADN endommagé par recombinaison non-homologue (non-homologous end-joining; NHEJ). DNA-PK est également impliquée dans de nombreux processus cellulaires autre que la réparation de l'ADN. Plusieurs travaux ont montré que les protéines impliquées dans la réparation des dommages de l'ADN tels que BRCA-1, MRN-11, PARP-1 et également de DNA-PK jouent un rôle important dans la métastase du cancer. Dans ce travail, nous nous sommes concentrées sur le rôle de DNA-PK dans les métastases du mélanome. Dans un premier temps, en utilisant les molécules Dbait 32Hc comme un moyen d'activer DNA-PK dans le noyau et le cytoplasme, nous avons identifié plusieurs nouvelles cibles cytoplasmiques de DNA-PK, dont la vimentine. Nous avons montré que DNA-PK phosphoryle la vimentine sur Ser459 et que cette forme phosphorylée est la plupart du temps située au niveau des protrusion cellulaires des cellules migratrices. Nous avons ensuite démontré que la vimentine-Ser459-P induite par le traitement de Dbait32Hc participe à l'inhibition de l'adhésion et la migration cellulaire. Ainsi, cette approche a conduit à l'identification de nouvelles cibles cytoplasmiques de DNA-PK et a révélé un lien entre la signalisation des dommages de l'ADN et le cytosquelette. Ensuite, nous avons montré que DNA-PK joue un rôle important dans la migration et invasion cellules en régulant la sécrétion des facteurs associés à la métastase. Nous avons montré que l'absence ou l’inhibition de DNA-PK conduit à une régulation négative des facteurs pro-métastatique sécrétés et à la régulation positive de facteurs anti-métastatiques sécrétés tels que les inhibiteurs des métalloprotéinases matricielles. Nous avons confirmé le rôle de DNA-PK in vivo dans l'implantation de la tumeur primaire et dans la formation des métastases. Ainsi, nos études ont évalué le rôle de DNA-PK sur le contrôle du microenvironnement de la tumeur par le contrôle de la sécrétion de facteurs importants pour la métastase. En résumé, nos résultats mettent en évidence l'importance de la DNA-PK comme cible de traitement anti-métastatique.The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase, which is a critical component of the DNA-damage repair pathways through non-homologous end-joining (NHEJ). Besides DNA repair, it is also involved in numerous cellular pathways. Emerging results show that proteins involved in DNA damage repair such as BRCA-1, MRN-11, PARP-1 and also DNA-PK could play a role in cancer metastasis. In the current study, we demonstrated the role of DNA-PK in melanoma metastasis. Firstly using Dbait 32Hc molecules as a tool for specifically activating DNA-PK in a nucleus and cytoplasm, we identified several new cytoplasmic targets of DNA-PK including vimentin. We established that DNA-PK phosphorylates vimentin on Ser459 and that this phosphorylation was mostly located at cell protrusions of melanoma migratory cells. Following this, we confirmed that vimentin-Ser459-P induced by Dbait 32Hc treatment participates to the inhibition of cell adhesion and migration. Thus, this approach led to the identification of downstream cytoplasmic targets of DNA-PK and revealed a connection between DNA damage signaling and the cytoskeleton. Secondly, we show that DNA-PK plays an important role in cell migration and melanoma cell invasion through the regulation of secretion of metastasis-associated factors. Absence or inhibition of DNA-PK leads to down-regulation of pro-metastatic secreted factors and up-regulation of anti-metastatic secreted factors such as inhibitors of matrix metalloproteinases. We confirmed in vivo, that DNA-PK is required for efficient primary tumor implantation and metastases formation. Thus, our studies demonstrate for the first time that DNA-PK acts on tumor microenvironment by controlling secretion of important factors for cell migration and invasion. In summary, our findings highlight the importance of DNA-PK as a target of anti-metastatic treatment

Significance of Streptococcus gallolyticus subsp. gallolyticus Association With Colorectal Cancer

Streptococcus gallolyticus subsp. gallolyticus Sgg (formerly known as S. bovis type I) is the main causative agent of septicemia and infective endocarditis (IE) in elderly and immunocompromised persons. It belongs to the few opportunistic bacteria, which have been strongly associated to colorectal cancer (CRC). A literature survey covering a period of 40 years (1970–2010) revealed that 65% of patients diagnosed with an invasive Sgg infection had a concomitant colorectal neoplasia. Sgg is associated mainly with early adenomas and may thus constitute an early marker for CRC screening. Sgg has been described as a normal inhabitant of the rumen of herbivores and in the digestive tract of birds. It is more rarely detected in human intestinal tract (2.5–15%). Recent molecular analyses indicate possible zoonotic transmission of Sgg. Thanks to the development of a genetic toolbox and to comparative genomics, a number of factors that are important for Sgg pathogenicity have been identified. This review will highlight the role of Sgg pili in host colonization and how their phase-variable expression contributes to mitigate the host immune responses and finally their use as serological diagnostic tool. We will then present experimental data addressing the core question whether Sgg is a cause or consequence of CRC. We will discuss a few recent studies examining the etiological versus non-etiological participation of Sgg in colorectal cancer with the underlying mechanisms

Apelin, APJ, and ELABELA : role in placental function, pregnancy, and foetal development - an overview

The apelinergic system, which includes the apelin receptor (APJ) as well as its two specific ligands, namely apelin and ELABELA (ELA/APELA/Toddler), have been the subject of many recent studies due to their pleiotropic effects in humans and other animals. Expression of these factors has been investigated in numerous tissues and organs—for example, the lungs, heart, uterus, and ovary. Moreover, a number of studies have been devoted to understanding the role of apelin and the entire apelinergic system in the most important processes in the body, starting from early stages of human life with regulation of placental function and the proper course of pregnancy. Disturbances in the balance of placental processes such as proliferation, apoptosis, angiogenesis, or hormone secretion may lead to specific pregnancy pathologies; therefore, there is a great need to search for substances that would help in their early diagnosis or treatment. A number of studies have indicated that compounds of the apelinergic system could serve this purpose. Hence, in this review, we summarized the most important reports about the role of apelin and the entire apelinergic system in the regulation of placental physiology and pregnancy

The anticancer peptide RT53 induces immunogenic cell death

<div><p>In recent years, immunogenic cell death (ICD) has emerged as a revolutionary concept in the development of novel anticancer therapies. This particular form of cell death is able, through the spatiotemporally defined emission of danger signals by the dying cell, to induce an effective antitumor immune response, allowing the immune system to recognize and eradicate malignant cells. To date, only a restricted number of chemotherapeutics can trigger ICD of cancer cells. We previously reported that a peptide, called RT53, spanning the heptad leucine repeat region of the survival protein AAC-11 fused to a penetrating sequence, selectively induces cancer cell death <i>in vitro</i> and <i>in vivo</i>. Interestingly, B16F10 melanoma cells treated by RT53 were able to mediate anticancer effects in a tumor vaccination model. Stimulated by this observation, we investigated whether RT53 might mediate ICD of cancer cells. Here, we report that RT53 treatment induces all the hallmarks of immunogenic cell death, as defined by the plasma membrane exposure of calreticulin, release of ATP and the exodus of high-mobility group box 1 protein (HMGB1) from dying cancer cells, through a non-regulated, membranolytic mode of action. In a prophylactic mouse model, vaccination with RT53-treated fibrosarcomas prevented tumor growth at the challenge site. Finally, local intratumoral injection of RT53 into established cancers led to tumor regression together with T-cell infiltration and the mounting of an inflammatory response in the treated animals. Collectively, our results strongly suggest that RT53 can induce <i>bona fide</i> ICD of cancer cells and illustrate its potential use as a novel antitumor and immunotherapeutic strategy.</p></div

RT53 triggers calreticulin exposure as well as the release of HMGB1 and ATP.

<p>(A) U2OS cells stably expressing CRT-GFP were treated with 10 μM of RT53 or 1 μM mitoxantrone for 6 h, in the presence or absence of zVAD-fmk. Cells were then, fixed, stained for DNA, and examined by fluorescence microscopy. (B) U2OS cells were left untreated or treated with 10 μM of RT53 or 200 nM thapsigargin (TG) as a positive control for 6 h. Cell lysates were analyzed by Western blot for phosphorylated and total protein eIF2α. (C) U2OS cells were left untreated or exposed to increasing concentrations of RT53 for 3h. Extracellular HMGB1 was then measured in the culture supernatant. Data are means±s.e.m. (<i>n</i> = 3). (D) U2OS cells were left untreated or exposed to increasing concentrations of RT53 for 3h. Extracellular ATP was then measured in the culture supernatant. Data are means±s.e.m. (<i>n</i> = 3).</p

Intratumoral administration of RT53 induces tumor necrosis, immune cells infiltration and inflammation.

<p>(A) Plasma was harvested 4, 24, 48 or 96 h following single intratumoral injection of normal saline (control) or 300 μg RT53 in normal saline and IL-1β (left panel) as well as IL-6 (right panel) levels were estimated using ELISA. Data from 3 animals are presented for each time point as mean±SEM. (B) Established MCA205 fibrosarcomas were surgically excised 24 h or 96 h post intratumoral injection with normal saline (control) or 300 μg RT53 in normal saline and sections subjected to H&E staining (top) or stained for CD3 or rabbit IgG isotype control (middle and bottom, respectively). (C) Number of infiltrating CD3-positive cells per view field following CD3 staining. Data from 3 animals are presented as mean±SEM. (D) Relative transcription of CCL2 and CXCL10 (normalized to GAPDH) as determined by real-time RT-PCR on samples of tumors 24 h after RT53 or normal saline injection. Values of CCL2 and CXCL10 are represented as fold change relative to untreated tumors, set to 1 (mean±SEM; n = 3).</p

RT53 induces unregulated necrotic cell death.

<p>(A) Ultrastructural analysis of RT53-mediated cell death. U2OS cells were left untreated (Control) or exposed to 15 μM of RT53 for 30 min. Cells were then analyzed by transmission electron microscopy following osmium tetroxide staining. (B) U2OS cells were exposed to 20 μM of RT53 in the presence or absence of 50 μM zVAD-fmk, 50 μM Necrostatin-1 (Nec-1) or 100 mM cyclosporin A (CsA) for 1 h. Necrotic cell death was monitored by lactate dehydrogenase (LDH) release from cells into the culture medium. The obtained values were normalized to those of the maximum LDH released (completely lysed) control. Data are means±s.e.m. (<i>n</i> = 3).</p

Efficient Therapeutic Delivery by a Novel Cell-Penetrating Peptide Derived from Acinus

International audienceIn this study, we have identified a novel cell-penetrating sequence, termed hAP10, from the C-terminus of the human protein Acinus. hAP10 was able to efficiently enter various normal and cancerous cells, likely through an endocytosis pathway, and to deliver an EGFP cargo to the cell interior. Cell penetration of a peptide, hAP10DR, derived from hAP10 by mutation of an aspartic acid residue to an arginine was dramatically increased. Interestingly, a peptide containing a portion of the heptad leucine repeat region domain of the survival protein AAC-11 (residues 377-399) fused to either hAP10 or hAP10DR was able to induce tumor cells, but not normal cells, death both ex vivo on Sézary patients' circulating cells and to inhibit tumor growth in vivo in a sub-cutaneous xenograft mouse model for the Sézary syndrome. Combined, our results indicate that hAP10 and hAP10DR may represent promising vehicles for the in vitro or in vivo delivery of bioactive cargos, with potential use in clinical settings