Autophagy induced by Helicobacter pylori infection is necessary for gastric cancer stem cell emergence

Background: The main cause of gastric cancer is the infection by the bacterium Helicobacter pylori which induces a chronic inflammation and an epithelial-to-mesenchymal transition (EMT) leading to the emergence of cells with cancer stem cell (CSC) properties. However, the underlying mechanisms have not been fully characterized. Moreover, H. pylori modulates the host cell autophagic process, but a few studies have investigated the role of this process in tumoral transformation. The aim of this study was to determine whether H. pylori-induced autophagy has a role in CSC emergence. Methods: Autophagic flux in response to H. pylori infection was characterized in AGS cell line expressing the tandem-tagged mCherry-GFP-LC3 protein and using a ratiometric flow cytometry analysis. Then, AGS and MKN45 cell lines were treated with bafilomycin or chloroquine, two pharmaceutical well-known inhibitors of autophagy, and different EMT and CSC characteristics were analyzed. Results: First, a co-expression of the gastric CSC marker CD44 and the autophagic marker LC3 in mice and human stomach tissues infected with H. pylori was observed. Then, we demonstrated in vitro that H. pylori was able to activate the autophagy process with a reduced autophagic flux. Finally, infected cells were treated with autophagy inhibitors, which reduced (i) appearance of mesenchymal phenotypes and migration ability related to EMT and (ii) CD44 expression as well as tumorsphere formation capacities reflecting CSC properties. Conclusion: In conclusion, all these data show that H. pylori-induced autophagy is implicated in gastric CSC emergence and could represent an interesting therapeutic target.This work was supported by the French foundation Ligue contre le Cancer (Pyrénées Atlantiques)

Cibler le cycle cellulaire : une nouvelle stratégie thérapeutique contre les cancers du sein déficients en ATIP3

Le cancer du sein est la première cause de décès par cancer chez la femme. Il est nécessaire de trouver des biomarqueurs permettant l'accès à des thérapies personnalisées, notamment pour les cancers du sein triple- négatifs (TNBC). ATIP3 est une protéine associée aux microtubules identifiée pour la première fois au sein de notre équipe. Une déficience en ATIP3 est retrouvée dans 65-80% des cancers TNBC et est associée à un mauvais pronostic clinique.Dans le but d'adresser le besoin d’une médecine personnalisée, mon projet de thèse a pour objectif l’identification de nouvelles approches thérapeutiques pour le traitement des cancers du sein déficients en ATIP3. En partant du fait que les cancers déficients en ATIP3 présentent un taux élevé d’aneuploïdie, nous avons émis l'hypothèse que l'augmentation de l'instabilité chromosomique avec des inhibiteurs de kinases du cycle cellulaire pourrait induire plus de mort cellulaire, rendant ainsi les tumeurs déficientes en ATIP3 plus vulnérables à une thérapie ciblée dirigée contre une kinase du cycle. J’ai mis en évidence que les cellules déficientes en ATIP3 sont plus sensibles à l'inhibition de la kinase WEE1, régulatrice de l'entre en mitose. Cette sensibilité accrue des cellules déficientes en ATIP3 à l’inhibition de WEE1 est due à une combinaison de effets : une induction d’un stress réplicatif et de dommages à l’ADN de manière excessive pendant la phase S du cycle, combinée à une entrée prématurée en mitose induisant une mortalité plus élevée. J’ai montré qu’en réponse à l’inhibition de WEE1 les cellules déficientes en ATIP3 présentent des mitoses aberrantes avec un détachement des protéines centromériques de l’ADN, qui lui-même est exclu du fuseau mitotique, empêchant ainsi la division et entrainant la mort cellulaire. J’ai également montré que ce phénotype aberrant est dû à l’activité dérégulée de l’hélicase/nucléase DNA2, qui fragmente l’ADN d’une manière excessive en réponse à l’inhibition de WEE1. Ainsi, l’ensemble de mes travaux propose la kinase WEE1 comme une nouvelle cible personnalisée contre les cancers du sein déficients en ATIP3, et décrit les effets de l’inhibition de WEE1 ainsi que les mécanismes par lesquels les cellules déficientes en ATIP3 sont plus sensibles à cette inhibition.Breast cancer is the leading cause of cancer- related deaths in women. The need to identify biomarkers for personalized therapies is imperative, especially for triple- negative breast cancers (TNBC). ATIP3 is a microtubule- associated protein identified by our team, and its deficiency is found in 65-80% of TNBC cases, associated with poor clinical prognosis. ATIP3-deficient tumors are of higher grade and exhibit a strong metastatic potential. My thesis project aims to identify novel therapeutic approaches for ATIP3-deficient breast cancer, driven by the urgent need for personalized medicine. Given that ATIP3-deficient cancers exhibit high levels of aneuploidy and chromosomal instability, we hypothesized that increasing genomic instability with cell cycle kinase inhibitors could induce more cell death, rendering ATIP3-deficient tumors more vulnerable to targeted kinase therapy. I have demonstrated that ATIP3-deficient cells are more sensitive to the inhibition of the WEE1 kinase, a regulator of mitotic entry. This heightened sensitivity results from a combination of effects, including induction of replicative stress and excessive DNA damage during the S phase of the cell cycle, in concert with premature mitotic entry and higher mortality. Furthermore, I have shown that in response to WEE1 inhibition, ATIP3-deficient cells exhibit aberrant mitoses with detachment of centromere proteins from DNA, preventing proper division and causing cell death. This abnormal phenotype is attributed to the dysregulated activity of the DNA2 helicase/nuclease, which excessively fragments DNA in response to WEE1 inhibition. In summary, my research highlights WEE1 kinase as a potential personalized target for ATIP3- deficient breast cancers and elucidates the effects of WEE1 inhibition and the mechanisms by which ATIP3- deficient cells become more susceptible to this inhibition

Microtubule-Associated Protein ATIP3, an Emerging Target for Personalized Medicine in Breast Cancer

International audienceBreast cancer is the leading cause of death by malignancy among women worldwide. Clinical data and molecular characteristics of breast tumors are essential to guide clinician’s therapeutic decisions. In the new era of precision medicine, that aims at personalizing the treatment for each patient, there is urgent need to identify robust companion biomarkers for new targeted therapies. This review focuses on ATIP3, a potent anti-cancer protein encoded by candidate tumor suppressor gene MTUS1, whose expression levels are markedly down-regulated in breast cancer. ATIP3 is a microtubule-associated protein identified both as a prognostic biomarker of patient survival and a predictive biomarker of breast tumors response to taxane-based chemotherapy. We present here recent studies pointing out ATIP3 as an emerging anti-cancer protein and a potential companion biomarker to be combined with future personalized therapy against ATIP3-deficient breast cancer

Microtubule-Associated Protein ATIP3, an Emerging Target for Personalized Medicine in Breast Cancer

Breast cancer is the leading cause of death by malignancy among women worldwide. Clinical data and molecular characteristics of breast tumors are essential to guide clinician’s therapeutic decisions. In the new era of precision medicine, that aims at personalizing the treatment for each patient, there is urgent need to identify robust companion biomarkers for new targeted therapies. This review focuses on ATIP3, a potent anti-cancer protein encoded by candidate tumor suppressor gene MTUS1, whose expression levels are markedly down-regulated in breast cancer. ATIP3 is a microtubule-associated protein identified both as a prognostic biomarker of patient survival and a predictive biomarker of breast tumors response to taxane-based chemotherapy. We present here recent studies pointing out ATIP3 as an emerging anti-cancer protein and a potential companion biomarker to be combined with future personalized therapy against ATIP3-deficient breast cancer

Organotypic Modeling of the Tumor Landscape

International audienceCancer is a complex disease and it is now clear that not only epithelial tumor cells play a role in carcinogenesis. The tumor microenvironment is composed of non-stromal cells, including endothelial cells, adipocytes, immune and nerve cells, and a stromal compartment composed of extracellular matrix, cancer-associated fibroblasts and mesenchymal cells. Tumorigenesis is a dynamic process with constant interactions occurring between the tumor cells and their surroundings. Even though all connections have not yet been discovered, it is now known that crosstalk between actors of the microenvironment drives cancer progression. Taking into account this complexity, it is important to develop relevant models to study carcinogenesis. Conventional 2D culture models fail to represent the entire tumor microenvironment properly and the use of animal models should be decreased with respect to the 3Rs rule. To this aim, in vitro organotypic models have been significantly developed these past few years. These models have different levels of complexity and allow the study of tumor cells alone or in interaction with the microenvironment actors during the multiple stages of carcinogenesis. This review depicts recent insights into organotypic modeling of the tumor and its microenvironment all throughout cancer progression. It offers an overview of the crosstalk between epithelial cancer cells and their microenvironment during the different phases of carcinogenesis, from the early cell autonomous events to the late metastatic stages. The advantages of 3D over classical 2D or in vivo models are presented as well as the most promising organotypic models. A particular focus is made on organotypic models used for studying cancer progression, from the less complex spheroids to the more sophisticated body-on-a-chip. Last but not least, we address the potential benefits of these models in personalized medicine which is undoubtedly a domain paving the path to new hopes in terms of cancer care and cure

Predicting and Overcoming Taxane Chemoresistance

International audienceTaxanes are microtubule-targeting drugs used as cytotoxic chemotherapy to treat most solid tumors. The development of resistance to taxanes is a major cause of therapeutic failure and overcoming chemoresistance remains an important challenge to improve patient's outcome. Extensive efforts have been made recently to identify predictive biomarkers to select populations of patients who will benefit from taxane-based chemotherapy and avoid inefficient treatment of patients with innate resistance. This, together with the discovery of new mechanisms of resistance that include metabolic reprogramming and dialogue between tumor and its microenvironment, pave the way to a new era of personalized medicine. In this review, we recapitulate recent insights into taxane resistance and present promising emerging strategies to overcome chemoresistance in the future

Detection of DNA damage by alkaline comet assay in mouse colonic mucosa

We recently characterized the association between DNA damage and immunoresponse in vivo in colonic mucosa of mice infected with a Salmonella Typhimurium strain expressing a genotoxin, known as typhoid toxin. In this protocol, we describe the specific steps for assessing DNA damage by the alkaline comet assay of colonic mucosal samples. The description of the comet assay protocol follows the international guidelines (Minimum Information for Reporting on the Comet Assay [Moller et al., 2020]). For complete details on the use and execution of this protocol, please refer to Martin et al. (2021)

Karakteristik vegetatif dan taksasi produksi kopi robusta tahun 2018 dan 2019 (Survey pada perkebunan kopi rakyat di Dusun Mandang, Desa Sucen, Kecamatan Gemawang, Kabupaten Temanggung)

This study aims to evaluate the dinamics of coffee production  in Mandang, Sucen Village, Gemawang District, Temanggung on 2018 and 2019. The research was carried out at  people coffee plantation in Mandang Hamlet, Sucen Village, Temanggung. Research using survey methods. Observation of performance with 30 samples taken by purposive sampling technique on 3 clones. Land suitability analysis was carried out at 3 observation points. The results obtained are: The vegetative characteristics  of robusta coffee BP 288 and BP 409 are better than  BP 358 clones, while the robusta coffee production is the same  on various clones and  plantation location.  The long dry season  in 2018 and 2019 has an effect on the decline of the number of leaves and coffee production in 2019 compared to 2018 in Mandang Hamlet, Sucen  Village, Gemawang district, Temanggung

The pro-apoptotic properties of a phytonutrient rich infusion of A. cherimola leaf extract on AML cells

Annonaceae family has broad uses in herbal medicine for treatment of several diseases, whether through seeds’ or leaves’ extracts. The present study investigates the antiproliferative and antitumor activity of Annona cherimola aqueous leaf (AAL) extract/infusion in acute myeloid leukemia (AML) cell lines in vitro. High-resolution LC-MS was first used to analyze the composition of the aqueous extract. Cell proliferation assay, Annexin V staining, cell cycle analysis, dual Annexin V/PI staining, cell death quantification by ELISA, ROS level detection and Western Blotting were then performed to elucidate the therapeutic effects of AAL extract. The results obtained revealed a potent antioxidant activity of AAL extract. Moreover, the extract exhibited dose- and time-dependent antiproliferative effects on AML cell lines by decreasing cell viability with an IC50 of 5.03% (v/v) at 24 h of treatment of KG-1 cells. This decrease in viability was accompanied with a significant increase in apoptotic cell death with cell cycle arrest and flipping of the phosphatidylserine from the inner to the outer leaflet of the cell membrane. The respective overexpression and downregulation of proapoptotic proteins like cleaved caspase-8, cleaved PARP-1 and Bax and antiapoptotic proteins like Bcl-2 further validated the apoptotic pathway induced by AAL on AML cells. Finally, LC-MS revealed the presence of several compounds like fatty acids, terpenes, phenolics, cinnamic acids and flavonoids that could contribute to the antioxidant and anti-cancer effects of this herbal infusion. In addition to the generally known nutritional effects of the Annona cherimola fruit and leaves, the presented data validates the antioxidant and anti-cancerous effects of the leaf infusion on AML cell lines, proposing its potential therapeutic use against acute myeloid leukemia with future in vivo and clinical trials

The Antioxidant and Proapoptotic Effects of Sternbergia clusiana Bulb Ethanolic Extract on Triple-Negative and Estrogen-Dependent Breast Cancer Cells In Vitro

Background: Sternbergia clusiana belongs to the Amaryllidaceae family and is recognized for the valuable biological activity of its major bioactive compounds. The aim of the current is to evaluate the anticancer effects of the ethanolic bulb extract of Sternbergia clusiana (ScBEE) on breast cancer cells in vitro and to further reveal the underlying cellular mechanism. Methods: An MTS cell viability assay was performed on MDA-MB-231 and MCF-7 cells, along with cell cycle analysis, cell death ELISA, Western blot analysis and an ROS production assay to decipher the mechanism of death. LC-MS/MS was also performed to identify the chemical composition of this ethanolic extract. Results: The results show a selective antiproliferative effect on both cell lines with no effect on normal mesenchymal stem cells. Further analysis suggested the activation of the apoptotic pathway as reflected by the increase in cellular and DNA fragmentation and alterations in apoptotic proteins such as Bax, Bcl-2 and c-PARP. ScBEE was also found to exhibit antioxidant effect, as shown by a decrease in ROS production. The underlying mechanism of action was explained by the presence of several bioactive compounds identified by LC-MS/MS, including alkaloids, terpenoids and phenols, which are elaborated in the manuscript. Conclusion: This study highlights the antioxidant and anticancerous properties of S.clusiana for breast cancer treatment