Determinants of Specific Binding of HMGB1 Protein to Hemicatenated DNA Loops

Protein HMGB1 has long been known as one of the most abundant non-histone proteins in the nucleus of mammalian cells, and has regained interest recently for its function as an extracellular cytokine. As a DNA-binding protein, HMGB1 facilitates DNA–protein interactions by increasing the flexibility of the double helix, and binds specifically to distorted DNA structures. We have previously observed that HMGB1 binds with extremely high affinity to a novel DNA structure, hemicatenated DNA loops (hcDNA), in which double-stranded DNA fragments containing a tract of poly(CA)·poly(TG) form a loop maintained at its base by a hemicatenane. Here, we show that the single HMGB1 domains A and B, the HMG-box domain of sex determination factor SRY, as well as the prokaryotic HMGB1-like protein HU, specifically interact with hcDNA (Kd0.5 nM). However, the affinity of full-length HMGB1 for hcDNA is three orders of magnitude higher (Kd<0.5 pM) and requires the simultaneous presence of both HMG-box domains A and B plus the acidic C-terminal tail on the molecule. Interestingly, the high affinity of the full-length protein for hcDNA does not decrease in the presence of magnesium. Experiments including a comparison of HMGB1 binding to hcDNA and to minicircles containing the CA/TG sequence, binding studies with HMGB1 mutated at intercalating amino acid residues (involved in recognition of distorted DNA structures), and exonuclease III footprinting, strongly suggest that the hemicatenane, not the DNA loop, is the main determinant of the affinity of HMGB1 for hcDNA. Experiments with supercoiled CA/TG-minicircles did not reveal any involvement of left-handed Z-DNA in HMGB1 binding. Our results point to a tight structural fit between HMGB1 and DNA hemicatenanes under physiological conditions, and suggest that one of the nuclear functions of HMGB1 could be linked to the possible presence of hemicatenanes in the cell

AMOTL1 Promotes Breast Cancer Progression and Is Antagonized by Merlin

AbstractThe Hippo signaling network is a key regulator of cell fate. In the recent years, it was shown that its implication in cancer goes well beyond the sole role of YAP transcriptional activity and its regulation by the canonical MST/LATS kinase cascade. Here we show that the motin family member AMOTL1 is an important effector of Hippo signaling in breast cancer. AMOTL1 connects Hippo signaling to tumor cell aggressiveness. We show that both canonical and noncanonical Hippo signaling modulates AMOTL1 levels. The tumor suppressor Merlin triggers AMOTL1 proteasomal degradation mediated by the NEDD family of ubiquitin ligases through direct interaction. In parallel, YAP stimulates AMOTL1 expression. The loss of Merlin expression and the induction of Yap activity that are frequently observed in breast cancers thus result in elevated AMOTL1 levels. AMOTL1 expression is sufficient to trigger tumor cell migration and stimulates proliferation by activating c-Src. In a large cohort of human breast tumors, we show that AMOTL1 protein levels are upregulated during cancer progression and that, importantly, the expression of AMOTL1 in lymph node metastasis appears predictive of the risk of relapse. Hence we uncover an important mechanism by which Hippo signaling promotes breast cancer progression by modulating the expression of AMOTL1

So close, yet so far : discrepancies between uveal and other melanomas. A Position Paper from UM Cure 2020

Despite much progress in our understanding of uveal melanoma (UM) over the past decades, this rare tumour is still often misclassified. Although UM, like other melanomas, is very probably derived from melanocytes, it is drastically different from cutaneous melanoma and most other melanoma subtypes in terms of epidemiology, aetiology, biology and clinical features, including an intriguing metastatic hepatotropism. UM carries distinctive prognostic chromosome alterations, somatic mutations and gene expression profiles, allowing an active tailored surveillance strategy and dedicated adjuvant clinical trials. There is no standard systemic treatment for disseminated UM at present. In contrast to cutaneous melanoma, UMs are not BRAF-mutated, thus curtailing the use of B-Raf inhibitors. Although these tumours are characterised by some immune infiltrates, immune checkpoint inhibitors are rarely effective, possibly due to a low mutation burden. UM patients across the world not only face rare cancer-related issues (e.g., specific management strategies, access to information and to expert centres), but also specific UM problems, which can be exacerbated by the common misconception that it is a subtype of cutaneous melanoma. As a European Consortium dedicated to research on UM and awareness on the disease, “UM Cure 2020” participants urge medical oncologists, pharmaceutical companies, and regulatory agencies to acknowledge UM as a melanoma with specific issues, in order to accelerate the development of new therapies for patients

Frailty is associated with in-hospital mortality in older hospitalised COVID-19 patients in the Netherlands:the COVID-OLD study

BACKGROUND: During the first wave of the coronavirus disease 2019 (COVID-19) pandemic, older patients had an increased risk of hospitalisation and death. Reports on the association of frailty with poor outcome have been conflicting. OBJECTIVE: The aim of the present study was to investigate the independent association between frailty and in-hospital mortality in older hospitalised COVID-19 patients in the Netherlands. METHODS: This was a multicentre retrospective cohort study in 15 hospitals in the Netherlands, including all patients aged ≥70 years, who were hospitalised with clinically confirmed COVID-19 between February and May 2020. Data were collected on demographics, co-morbidity, disease severity and Clinical Frailty Scale (CFS). Primary outcome was in-hospital mortality. RESULTS: A total of 1,376 patients were included (median age 78 years (interquartile range 74-84), 60% male). In total, 499 (38%) patients died during hospital admission. Parameters indicating presence of frailty (CFS 6-9) were associated with more co-morbidities, shorter symptom duration upon presentation (median 4 versus 7 days), lower oxygen demand and lower levels of C-reactive protein. In multivariable analyses, the CFS was independently associated with in-hospital mortality: compared with patients with CFS 1-3, patients with CFS 4-5 had a two times higher risk (odds ratio (OR) 2.0 (95% confidence interval (CI) 1.3-3.0)) and patients with CFS 6-9 had a three times higher risk of in-hospital mortality (OR 2.8 (95% CI 1.8-4.3)). CONCLUSIONS: The in-hospital mortality of older hospitalised COVID-19 patients in the Netherlands was 38%. Frailty was independently associated with higher in-hospital mortality, even though COVID-19 patients with frailty presented earlier to the hospital with less severe symptoms

Association of the PHACTR1/EDN1 genetic locus with spontaneous coronary artery dissection

Background: Spontaneous coronary artery dissection (SCAD) is an increasingly recognized cause of acute coronary syndromes (ACS) afflicting predominantly younger to middle-aged women. Observational studies have reported a high prevalence of extracoronary vascular anomalies, especially fibromuscular dysplasia (FMD) and a low prevalence of coincidental cases of atherosclerosis. PHACTR1/EDN1 is a genetic risk locus for several vascular diseases, including FMD and coronary artery disease, with the putative causal noncoding variant at the rs9349379 locus acting as a potential enhancer for the endothelin-1 (EDN1) gene. Objectives: This study sought to test the association between the rs9349379 genotype and SCAD. Methods: Results from case control studies from France, United Kingdom, United States, and Australia were analyzed to test the association with SCAD risk, including age at first event, pregnancy-associated SCAD (P-SCAD), and recurrent SCAD. Results: The previously reported risk allele for FMD (rs9349379-A) was associated with a higher risk of SCAD in all studies. In a meta-analysis of 1,055 SCAD patients and 7,190 controls, the odds ratio (OR) was 1.67 (95% confidence interval [CI]: 1.50 to 1.86) per copy of rs9349379-A. In a subset of 491 SCAD patients, the OR estimate was found to be higher for the association with SCAD in patients without FMD (OR: 1.89; 95% CI: 1.53 to 2.33) than in SCAD cases with FMD (OR: 1.60; 95% CI: 1.28 to 1.99). There was no effect of genotype on age at first event, P-SCAD, or recurrence. Conclusions: The first genetic risk factor for SCAD was identified in the largest study conducted to date for this condition. This genetic link may contribute to the clinical overlap between SCAD and FMD

Facteurs d'assemblage de la chromatine et organisation de l'hétérochromatine du normal au pathologique

In cancer cells, the organization of DNA in chromatin is frequently affected. Thus, it is crucial to understand how factors involved in chromatin organization contribute to tumorigenesis. Of particular interest in this context is the Chromatin Assembly Factor 1 (CAF-1). CAF-1 is a complex involved in chromatin assembly coupled to DNA synthesis, both during DNA replication and DNA repair. Two subunits of the CAF-1 complex are downregulated in quiescent cells and can be used as proliferation markers in cancer cells. In addition, CAF-1 has a role in the maintenance of the compact chromatin domains near the centromeres, called pericentric heterochromatin, through its interaction with Heterochromatin Protein 1 (HP1). Three HP1 isoforms (HP1α, β et γ) exist in mammalian cells, among which HP1α associates most specifically with pericentric heterochromatin regions. During my PhD, I have addressed two main questions: First, how do the three human HP1 isoforms relate to cell proliferation and tumorigenesis? Using both cell line models and tumoral and healthy human tissue samples, I showed that the expression of HP1α isoform, but not HP1β ou γ, is proliferation-dependent. Downregulation of HP1α, specifically, results in mitotic defects. In addition, HP1α, but not HP1β ou γ, is overexpressed in several tumoral tissues compared to the corresponding healthy tissues. In breast cancer, the level of HP1α overexpression significantly correlates with global survival and the occurence of metastasis. Our results suggest that HP1α overexpression confers a growth advantage to tumor cells that is related to the organization of pericentric heterochromatin and the passage of mitosis. We put forward HP1α as new prognostic marker in breast cancer and potentially other cancers. This study gave rise to the deposition of a patent and a publication in EMBO Molecular Medecine. The second main question I have addressed is: how do quiescent cells, which express CAF-1 at very low levels, deal with chromatin assembly coupled to DNA repair? This question is of particular interest since a differential repair capacity between tumoral (proliferative) cells and healthy (quiescent) cells will affect the efficiency and the toxicity of genotoxic cancer treatments, such as chemo- and radiotherapy Using ultra-violet (UV) irradiation as a means to induce DNA lesions, I was able to show that CAF-1 expression is not induced upon DNA damage and that the low levels of CAF-1 are sufficient for its recruitment to sites of UV lesions, suggesting that its function is conserved in quiescence. However, we observe a delayed repair of a specific type of UV lesions in quiescence. Our results suggest that the repair of these lesions might require additional chromatin dynamics, which would be the limiting step in quiescence, potentially due to the extremely low levels of CAF-1. These observations gave rise to a manuscript that is currently in preparation. In these two major projects, I have demonstrated how factors involved in chromatin organization are related to cell proliferation, tumorigenesis and, more generally, genome stability. In addition, we have been able to put forward a new marker of clinical interest for breast cancer prognosis.Dans les cellules cancéreuses, des défauts affectant l'organisation d'ADN en chromatine sont fréquemment observés. L'étude de facteurs impliqués dans cette organisation est donc essentielle pour mieux appréhender leur implication dans la tumorigénèse. Un facteur particulièrement intéressant dans ce contexte est le facteur d'assemblage de la chromatine, le complexe CAF-1 (Chromatin Assembly Factor 1). CAF-1 est impliqué dans l'assemblage en chromatine de l'ADN lors de la réplication et la réparation de l'ADN. Deux sous-unités de CAF-1 sont sous-exprimés dans les cellules non-proliférantes (quiescentes) et constituent des marqueurs de prolifération dans le cancer. De plus, CAF-1 a un rôle au niveau des régions de chromatine dense proches des centromères, l'hétérochromatine péricentrique, par son interaction avec les protéines HP1 (Heterochromatin Protein 1). Il existe trois isoformes de HP1 dans les cellules mammaires (HP1α, β et γ), dont HP1α est le plus spécifiquement associé aux régions d'hétérochromatine péricentrique, impliqués dans la répression des gènes et la ségrégation des chromosomes. Pendant ma thèse, je me suis penchée sur deux questions majeures: Premièrement, est- ce que l'expression des isoformes de HP1 est régulée d'une façon dépendante de la prolifération et de la tumorigénèse ? En combinant des modèles de lignées cellulaires et des échantillons de tissu humain, j'ai pu montrer que l'expression de l'isoforme HP1α, mais pas HP1β ou γ, est dépendante de la prolifération. La déplétion de HP1α, spécifiquement, affecte le passage de la mitose. De plus, HP1α, mais pas HP1β ou γ, est surexprimé dans de nombreux types de cancer comparé aux tissus sains correspondants. La surexpression de HP1α dans le cancer du sein est corrélée de façon significative à la survie des patientes et la formation de métastases. Ces résultats révèlent HP1α comme un marqueur pronostique dans le cancer du sein et potentiellement dans d'autres types de cancer. Nous proposons que la surexpression de HP1α présente un avantage sélectif pour les cellules cancéreuses, liée à l'organisation de l'hétérochromatine péricentrique et le passage de la mitose. Ces résultats ont donné lieu à un brevet et à une publication dans EMBO Molecular Medecine. La seconde question à laquelle je me suis intéressée est : comment des cellules quiescentes, qui expriment peu de CAF-1, gèrent l'assemblage de la chromatine couplé à la réparation de l'ADN ? En effet, une capacité de réparation différente entre cellules proliférantes (tumorales) et quiescentes (saines) aura un impact majeur sur l'efficacité et la toxicité des traitements génotoxiques comme la chimio- et la radiothérapie. J'ai pu montrer que, dans les cellules quiescentes, les irradiations aux ultra-violets (UV) n'induisent pas l'expression de CAF-1. De plus, la faible quantité de CAF-1 est recrutée aux sites des lésions d'UV, suggérant que sa fonction dans la réparation est conservée hors du cycle cellulaire. Cependant, en quiescence, nous observons une réparation retardée d'un type spécifique de lésions, qui reflète potentiellement une difficulté des cellules quiescentes à gérer la prise en charge de ces lésions au sein de la chromatine. Ces résultats font l'objet d'un manuscrit actuellement en préparation. Dans ces deux projets majeurs, j'ai mis en avant comment des facteurs de l'organisation de la chromatine peuvent être impliqués dans la prolifération, la tumorigénèse et la réparation d'ADN suite à des traitements génotoxiques. De plus, nous avons pu proposer un nouvel outil d'intérêt médical pour le pronostique des cancer du sein

PARP Inhibition Increases the Response to Chemotherapy in Uveal Melanoma

Uveal melanoma (UM) remains without effective therapy at the metastatic stage, which is associated with BAP-1 (BRCA1 associated protein) mutations. However, no data on DNA repair capacities in UM are available. Here, we use UM patient-derived xenografts (PDXs) to study the therapeutic activity of the PARP inhibitor olaparib, alone or in combination. First, we show that the expression and the activity of PARP proteins is similar between the PDXs and the corresponding patient&rsquo;s tumors. In vivo experiments in the PDX models showed that olaparib was not efficient alone, but significantly increased the efficacy of dacarbazine. Finally, using reverse phase protein arrays and immunohistochemistry, we identified proteins involved in DNA repair and apoptosis as potential biomarkers predicting response to the combination of olaparib and dacarbazine. We also observed a high increase of phosphorylated YAP and TAZ proteins after dacarbazine + olaparib treatment. Our results suggest that PARP inhibition in combination with the alkylating agent dacarbazine could be of clinical interest for UM treatment. We also observe an interesting effect of dacarbazine on the Hippo pathway, confirming the importance of this pathway in UM