Chloroquine plays a cell-dependent role in the response to treatment of pancreatic adenocarcinoma

In this study, our aim is to assess the role played by autophagy and its inhibition in the different PDAC cellular compartments, and its involvement in chemo-resistance using primary human pancreatic cancer-derived cells (PCC) and Cancer Associated Fibroblasts (CAF). Autophagy flux, as measured by LC3-I and -II in the presence of Chloroquine, showed a variable level in PCC and CAFs. We found no correlation between autophagy level and degree of tumor differentiation. Association of Chloroquine with gemcitabine, 5FU, oxaliplatin, irinotecan and docetaxel revealed that its effect on survival is cell- and drug-dependent in vitro and in vivo. In addition, we demonstrated that autophagy in CAFs can play an important role in sensitizing PDAC to anticancer treatments since its inhibition increased the resistance of PCCs to gemcitabine. In conclusion, this work clearly shows a heterogeneity in the effect of Chloroquine and highlights a role of CAFs autophagy in sensitizing tumors to treatments. It also reveals that the role of autophagy is more complex than expected in PDAC as well as its sensitivity to treatments.Fil: Molejon, Maria Ines. Institut National de la Santé et de la Recherche Médicale; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; ArgentinaFil: Swayden, Mirna. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Fanale, Daniele. 3 Department of Surgical, Oncological and Oral Sciences, Section of Medical Oncology, University of Palermo, 90127 Palermo, Italy.; Argentina. University of Palermo. Department of Surgical, Oncological and Oral Sciences; ItaliaFil: Bintz, Jennifer. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Gayet, Odile. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Soubeyran, Philippe. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Iovanna, Juan Lucio. Institut National de la Santé et de la Recherche Médicale; Franci

Blocking Nupr1 Protein, A Successful Approach for Pancreatic Adenocarcinoma Treatment

Background: Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic malignancy. Nuclear protein 1 (NUPR1) is a recognized protein, over-expressed and involved in PDAC development. NUPR1 belongs to the special class of intrinsically disordered proteins (IDPs) and it is implicated in cell signaling and regulatory functions. The multifunctional nature of NUPR1 renders it as an attractive target for drug design and development. Aim: Identify a small molecule inhibiting protein-protein interactions in NUPR1 and able to interfere with any of NUPR1 key oncogenic activities, thus, constituting a new chemotherapy strategy against PDAC. Methods: Ligand-induced stabilization against thermal denaturation (thermal-shift assay) was employed for identifying potential NUPR1-interacting compounds. An in vitro molecular screening based on thermal denaturation of NUPR1 in the presence of a variety of potential ligands was performed using a collection of 1120 compounds. All compounds are FDA-approved drugs for different therapeutic indications, exhibiting high chemical and pharmacological diversity, as well as good bioavailability and safety parameters in humans. Direct interaction of selected compounds with NUPR1 was assessed experimentally (calorimetry, fluorescence spectroscopy, nuclear magnetic resonance, and proximity ligation assay) and computationally (molecular dynamics simulations). Compound efficacy was determined in PDAC-derived cell-based assays and in vivo assays on xenografted PDACderived cells in mice. Comparisons of treatment outcome were tested for statistical significance by using the t-test, and statistical significance was assumed at a p-value lower than 0.05. Results: Fifteen candidates were selected, and their interactions with NUPR1 were characterized. In vitro experiments with MiaPaCa-2 cells treated using 10 µM of compounds for 6 days showed that two of the compounds (C13 and C15) were very efficient in diminishing cell viability (10 ± 3% and 26 ± 7%, respectively; assays in triplicates (n = ) p= 0.01). These values were similar to those obtained with oxaliplatin (10 ± 2%; p= 0.01). Also, they reduced cell migration (from 10-20% wound-healing ability compared to 50% in control assays; p= 0.05) and colony formation (completely suppressed in the presence of both compounds; p= 0.01). In addition, the most promising compound, C15, interfered with the interaction of NUPR1 with MSL1, one of the NUPR1 binding partners (Figure 1). The administration of a 10 mg/Kg dose of C15 promoted complete arrest of tumor development on xenografted PDAC-derived cells in mice (Figure 2). Conclusion: We report the discovery of a compound specifically active against PDAC and interfering with NUPR1. In addition, we demonstrate that it is possible to identify small molecules able to modulate the function of complex targets such as IDPs

Functional Characterization of Nupr1L, A Novel p53-Regulated Isoform of the High-Mobility Group (HMG)-Related Protumoral Protein Nupr1

We have previously demonstrated a crucial role of nuclear protein 1 (NUPR1) in tumor development and progression. In this work, we report the functional characterization of a novel Nupr1-like isoform (NUPR1L) and its functional interaction with the protumoral factor NUPR1. Through the use of primary sequence analysis, threading, and homology-based molecular modeling, as well as expression and immunolocalization, studies reveal that NUPR1L displays properties, which are similar to member of the HMG-like family of chromatin regulators, including its ability to translocate to the cell nucleus and bind to DNA. Analysis of the NUPR1L promoter showed the presence of two p53-response elements at positions -37 and -7, respectively. Experiments using reporter assays combined with site-directed mutagenesis and using cells with controllable p53 expression demonstrate that both of these sequences are responsible for the regulation of NUPR1L expression by p53. Congruently, NUPR1L gene expression is activated in response to DNA damage induced by oxaliplatin treatment or cell cycle arrest induced by serum starvation, two well-validated methods to achieve p53 activation. Interestingly, expression of NUPR1L downregulates the expression of NUPR1, its closely related protumoral isoform, by a mechanism that involves the inhibition of its promoter activity. At the cellular level, overexpression of NUPR1L induces G1 cell cycle arrest and a decrease in their cell viability, an effect that is mediated, at least in part, by downregulating NUPR1 expression. Combined, these experiments constitute the first functional characterization of NUPR1L as a new p53-induced gene, which negatively regulates the protumoral factor NUPR1.Fil: Lopez, Maria Belen. Centre de Recherche En Cancerologie de Marseille; FranciaFil: Garcia, Maria Noé. Centre de Recherche En Cancerologie de Marseille; FranciaFil: Grasso, Daniel Hector. Centre de Recherche En Cancerologie de Marseille; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bintz, Jennifer. Centre de Recherche En Cancerologie de Marseille; FranciaFil: Molejon, Maria Ines. Centre de Recherche En Cancerologie de Marseille; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Velez, Gabriel. Mayo Clinic; Estados UnidosFil: Lomberk, Gwen. Mayo Clinic; Estados UnidosFil: Neira, Jose Luis. Universidad de Miguel Hernández; EspañaFil: Urrutia, Raul. Mayo Clinic; Estados UnidosFil: Iovanna, Juan. Centre de Recherche En Cancerologie de Marseille; Franci

An Action-Oriented AI Policy Toolkit for Technology Audits by Community Advocates and Activists

Motivated by the extensive documented disparate harms of artificial intelligence (AI), many recent practitioner-facing reflective tools have been created to promote responsible AI development. However, the use of such tools internally by technology development firms addresses responsible AI as an issue of closed-door compliance rather than a matter of public concern. Recent advocate and activist efforts intervene in AI as a public policy problem, inciting a growing number of cities to pass bans or other ordinances on AI and surveillance technologies. In support of this broader ecology of political actors, we present a set of reflective tools intended to increase public participation in technology advocacy for AI policy action. To this end, the Algorithmic Equity Toolkit (the AEKit) provides a practical policy-facing definition of AI, a flowchart for assessing technologies against that definition, a worksheet for decomposing AI systems into constituent parts, and a list of probing questions that can be posed to vendors, policy-makers, or government agencies. The AEKit carries an action-orientation towards political encounters between community groups in the public and their representatives, opening up the work of AI reflection and remediation to multiple points of intervention. Unlike current reflective tools available to practitioners, our toolkit carries with it a politics of community participation and activism

Measuring the availability of human resources for health and its relationship to universal health coverage for 204 countries and territories from 1990 to 2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: Human resources for health (HRH) include a range of occupations that aim to promote or improve human health. The UN Sustainable Development Goals (SDGs) and the WHO Health Workforce 2030 strategy have drawn attention to the importance of HRH for achieving policy priorities such as universal health coverage (UHC). Although previous research has found substantial global disparities in HRH, the absence of comparable cross-national estimates of existing workforces has hindered efforts to quantify workforce requirements to meet health system goals. We aimed to use comparable and standardised data sources to estimate HRH densities globally, and to examine the relationship between a subset of HRH cadres and UHC effective coverage performance. Methods: Through the International Labour Organization and Global Health Data Exchange databases, we identified 1404 country-years of data from labour force surveys and 69 country-years of census data, with detailed microdata on health-related employment. From the WHO National Health Workforce Accounts, we identified 2950 country-years of data. We mapped data from all occupational coding systems to the International Standard Classification of Occupations 1988 (ISCO-88), allowing for standardised estimation of densities for 16 categories of health workers across the full time series. Using data from 1990 to 2019 for 196 of 204 countries and territories, covering seven Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) super-regions and 21 regions, we applied spatiotemporal Gaussian process regression (ST-GPR) to model HRH densities from 1990 to 2019 for all countries and territories. We used stochastic frontier meta-regression to model the relationship between the UHC effective coverage index and densities for the four categories of health workers enumerated in SDG indicator 3.c.1 pertaining to HRH: physicians, nurses and midwives, dentistry personnel, and pharmaceutical personnel. We identified minimum workforce density thresholds required to meet a specified target of 80 out of 100 on the UHC effective coverage index, and quantified national shortages with respect to those minimum thresholds. Findings: We estimated that, in 2019, the world had 104·0 million (95% uncertainty interval 83·5–128·0) health workers, including 12·8 million (9·7–16·6) physicians, 29·8 million (23·3–37·7) nurses and midwives, 4·6 million (3·6–6·0) dentistry personnel, and 5·2 million (4·0–6·7) pharmaceutical personnel. We calculated a global physician density of 16·7 (12·6–21·6) per 10 000 population, and a nurse and midwife density of 38·6 (30·1–48·8) per 10 000 population. We found the GBD super-regions of sub-Saharan Africa, south Asia, and north Africa and the Middle East had the lowest HRH densities. To reach 80 out of 100 on the UHC effective coverage index, we estimated that, per 10 000 population, at least 20·7 physicians, 70·6 nurses and midwives, 8·2 dentistry personnel, and 9·4 pharmaceutical personnel would be needed. In total, the 2019 national health workforces fell short of these minimum thresholds by 6·4 million physicians, 30·6 million nurses and midwives, 3·3 million dentistry personnel, and 2·9 million pharmaceutical personnel. Interpretation: Considerable expansion of the world's health workforce is needed to achieve high levels of UHC effective coverage. The largest shortages are in low-income settings, highlighting the need for increased financing and coordination to train, employ, and retain human resources in the health sector. Actual HRH shortages might be larger than estimated because minimum thresholds for each cadre of health workers are benchmarked on health systems that most efficiently translate human resources into UHC attainment

Expression of POU2F3 Transcription Factor Control Inflammation, Immunological Recruitment and Metastasis of Pancreatic Cancer in Mice

TUFT cells have been described as strong modulators of inflammatory cells in several tissues including pancreas. TUFT cells, also known as DCLK1+ cells, are dependent of the transcriptional factor POU2F3. Several works report DCLK1+ cells in early stages of PDAC development suggesting an important role of TUFT cells in PDAC development. Therefore, we developed a mice model (PDX1-Cre;KrasG12D;Ink4afl/fl), known as PKI model, deficient or not of POU2F3. In this animal model, deficiency of POU2F3 results in the absence of TUFT cells in PDAC as expected. Although, tumor development and growth are not significantly influenced, the development of liver metastasis was almost completely inhibited in POU2F3-deficient mice. Surprisingly, the absence of metastasis was associated with a higher expression of epithelial-to-mesenchymal transition markers, but to a lower inflammatory microenvironment suggesting that inflammation influences metastasis production more than epithelial-to-mesenchymal transition in this animal model. We can conclude that POU2F3 could be a new therapeutic target for control PDAC progression

Impact of Notch/HES1 pathway activation on the transformed phenotype of pancreatic cancer cells

Résumé: La réactivation de la voie Notch est une des caractéristiques les plus redondantes observées au cours de la carcinogenèse pancréatique. L’expression aberrante de la protéine HES1, cible de la signalisation Notch, est observée dès les premières phases de la carcinogenèse (Miyamoto et al., 2003). L’utilisation d’inhibiteurs de gamma-sécrétase semble être une stratégie thérapeutique prometteuse soutenue par de nombreuses études in vitro et in vivo (Plentz et al. 2009). Cette étude vise à démontrer que l’inhibition de la voie Notch et particulièrement de sa protéine cible, HES1, permet de réduire les propriétés tumorigéniques des cellules cancéreuses pancréatiques humaines. Pour cela, nous avons utilisé deux lignées cellulaires issues d’adénocarcinomes pancréatiques humains : MIAPaCa-2 et BxPC-3 ; celles-ci ont été traitées avec des inhibiteurs de gamma-sécrétase et infectées par des lentivirus codant pour des shARNs ciblant de manière spécifique les ARNm de Notch1 ou Hes1. Nos RÉSULTATS démontrent que les cellules BxPC-3 présentent un plus fort niveau d'activation de NOTCH1 qui corrèle avec un plus fort niveau d'expression d’HES1 comparativement aux cellules MIAPaCa-2. L’inhibition de la voie Notch réduit la prolifération des cellules BxPC-3 sans affecter celle des cellules MIAPaCa-2 ni même la survie cellulaire des deux lignées. De plus, on observe une diminution de la migration des cellules BxPC-3 suite à l’inhibition de la voie Notch. Notre étude suggère, d'une part, que l’efficacité des inhibiteurs de gamma-secrétase ne dépend pas seulement de l’expression des récepteurs NOTCHs mais principalement de leur niveau d'activation. Et, d’autre part, une implication directe et majeure de la protéine cible HES1 dans la médiation des effets de la voie Notch.||Abstract

Impact of Notch/HES1 pathway activation on the transformed phenotype of pancreatic cancer cells

Résumé: La réactivation de la voie Notch est une des caractéristiques les plus redondantes observées au cours de la carcinogenèse pancréatique. L’expression aberrante de la protéine HES1, cible de la signalisation Notch, est observée dès les premières phases de la carcinogenèse (Miyamoto et al., 2003). L’utilisation d’inhibiteurs de gamma-sécrétase semble être une stratégie thérapeutique prometteuse soutenue par de nombreuses études in vitro et in vivo (Plentz et al. 2009). Cette étude vise à démontrer que l’inhibition de la voie Notch et particulièrement de sa protéine cible, HES1, permet de réduire les propriétés tumorigéniques des cellules cancéreuses pancréatiques humaines. Pour cela, nous avons utilisé deux lignées cellulaires issues d’adénocarcinomes pancréatiques humains : MIAPaCa-2 et BxPC-3 ; celles-ci ont été traitées avec des inhibiteurs de gamma-sécrétase et infectées par des lentivirus codant pour des shARNs ciblant de manière spécifique les ARNm de Notch1 ou Hes1. Nos RÉSULTATS démontrent que les cellules BxPC-3 présentent un plus fort niveau d'activation de NOTCH1 qui corrèle avec un plus fort niveau d'expression d’HES1 comparativement aux cellules MIAPaCa-2. L’inhibition de la voie Notch réduit la prolifération des cellules BxPC-3 sans affecter celle des cellules MIAPaCa-2 ni même la survie cellulaire des deux lignées. De plus, on observe une diminution de la migration des cellules BxPC-3 suite à l’inhibition de la voie Notch. Notre étude suggère, d'une part, que l’efficacité des inhibiteurs de gamma-secrétase ne dépend pas seulement de l’expression des récepteurs NOTCHs mais principalement de leur niveau d'activation. Et, d’autre part, une implication directe et majeure de la protéine cible HES1 dans la médiation des effets de la voie Notch.||Abstract

Expression of POU2F3 Transcription Factor Control Inflammation, Immunological Recruitment and Metastasis of Pancreatic Cancer in Mice

International audienceTUFT cells have been described as strong modulators of inflammatory cells in several tissues including pancreas. TUFT cells, also known as DCLK1+ cells, are dependent of the transcriptional factor POU2F3. Several works report DCLK1+ cells in early stages of PDAC development suggesting an important role of TUFT cells in PDAC development. Therefore, we developed a mice model (PDX1-Cre;KrasG12D;Ink4afl/fl), known as PKI model, deficient or not of POU2F3. In this animal model, deficiency of POU2F3 results in the absence of TUFT cells in PDAC as expected. Although, tumor development and growth are not significantly influenced, the development of liver metastasis was almost completely inhibited in POU2F3-deficient mice. Surprisingly, the absence of metastasis was associated with a higher expression of epithelial-to-mesenchymal transition markers, but to a lower inflammatory microenvironment suggesting that inflammation influences metastasis production more than epithelial-to-mesenchymal transition in this animal model. We can conclude that POU2F3 could be a new therapeutic target for control PDAC progression

Combined AURKA and H3K9 Methyltransferase Targeting Inhibits Cell Growth By Inducing Mitotic Catastrophe

International audienceThe current integrative pathobiologic hypothesis states that pancreatic cancer (PDAC) develops and progresses in response to an interaction between known oncogenes and downstream epigenomic regulators. Congruently, this study tests a new com-binatorial therapy based on the inhibition of the Aurora kinase A (AURKA) oncogene and one of its targets, the H3K9 methylation– based epigenetic pathway. This therapeutic combination is effective at inhibiting the in vitro growth of PDAC cells both, in monolayer culture systems, and in three-dimensional spheroids and organoids. The combination also reduces the growth of PDAC xenografts in vivo. Mechanistically, it was found that inhibiting methyltransferases of the H3K9 pathway in cells, which are arrested in G 2 –M after targeting AURKA, decreases H3K9 meth-ylation at centromeres, induces mitotic aberrations, triggers an aberrant mitotic check point response, and ultimately leads to mitotic catastrophe. Combined, these data describe for the first time a hypothesis-driven design of an efficient combinatorial treatment that targets a dual oncogenic–epigenomic pathway to inhibit PDAC cell growth via a cytotoxic mechanism that involves perturbation of normal mitotic progression to end in mitotic catastrophe. Therefore, this new knowledge has significant mech-anistic value as it relates to the development of new therapies as well as biomedical relevance. Implications: These results outline a model for the combined inhibition of a genetic-to-epigenetic pathway to inhibit cell growth and suggest an important and provocative consideration for harnessing the capacity of cell-cycle inhibitors to enhance the future use of epigenetic inhibitors