Acetyl-CoA carboxylase 1 controls a lipid droplet-peroxisome axis and is a vulnerability of endocrine-resistant ER+ breast cancer.

Targeting aromatase deprives ER+ breast cancers of estrogens and is an effective therapeutic approach for these tumors. However, drug resistance is an unmet clinical need. Lipidomic analysis of long-term estrogen-deprived (LTED) ER+ breast cancer cells, a model of aromatase inhibitor resistance, revealed enhanced intracellular lipid storage. Functional metabolic analysis showed that lipid droplets together with peroxisomes, which we showed to be enriched and active in the LTED cells, controlled redox homeostasis and conferred metabolic adaptability to the resistant tumors. This reprogramming was controlled by acetyl-CoA-carboxylase-1 (ACC1), whose targeting selectively impaired LTED survival. However, the addition of branched- and very long-chain fatty acids reverted ACC1 inhibition, a process that was mediated by peroxisome function and redox homeostasis. The therapeutic relevance of these findings was validated in aromatase inhibitor-treated patient-derived samples. Last, targeting ACC1 reduced tumor growth of resistant patient-derived xenografts, thus identifying a targetable hub to combat the acquisition of estrogen independence in ER+ breast cancers.

The D647N mutation of FGFR1 induces ligand-independent receptor activation.

The activation loop (A-loop) of kinases, a key regulatory region, is recurrently mutated in several kinase proteins in cancer resulting in dysregulated kinase activity and response to kinase inhibitors. FGFR1 receptor tyrosine kinase represents an important oncogene and therapeutic target for solid and hematological tumors. Here we investigate the biochemical and molecular effects of D647N mutation lying in the A-loop of FGFR1. When expressed in normal and tumoral in vitro cell models, FGFR1D647N is phosphorylated also in the absence of ligands, and this is accompanied by the activation of intracellular signaling. The expression of FGFR1D647N significantly increases single and collective migration of cancer cells in vitro and in vivo, when compared to FGFR1WT. FGFR1D647N expression exacerbates the aggressiveness of cancer cells, increasing their invasiveness in vitro and augmenting their pro-angiogenic capacity in vivo. Remarkably, the D647N mutation significantly increases the sensitivity of FGFR1 to the ATP-competitive inhibitor Erdafitinib suggesting the possibility that this mutation could become a specific target for the development of new inhibitors. Although further efforts are warranted for an exhaustive description of the activation mechanisms, for the identification of more specific inhibitors and for confirming the clinical significance of mutated FGFR1D647N, overall our data demonstrate that the D647N substitution of FGFR1 is a novel pro-oncogenic activating mutation of the receptor that, when found in cancer patients, may anticipate good response to erdafitinib treatment.

Alternative In Vivo Models to Study Teratoma.

Embryonic stem cells give rise to teratomas when injected in vivo in experimental animal models. The characterization, the manipulation, and the breaking off of this specific characteristic are doubtlessly the last frontier for the applications of stem cells in translational medicine. Moreover, the urgency to adapt to new scientific demands drives the researcher to find alternative and faster models for testing the teratogenic properties of embryonic stem cells. Here, we compare the emerging model of the chick embryo chorioallantoic membrane (CAM) to the murine model, which represents the gold standard procedure for teratogenesis.

The impact of adipokines on vascular networks in adipose tissue.

Adipose tissue (AT) is a highly active and plastic endocrine organ. It secretes numerous soluble molecules known as adipokines, which act locally to AT control the remodel and homeostasis or exert pleiotropic functions in different peripheral organs. Aberrant production or loss of certain adipokines contributes to AT dysfunction associated with metabolic disorders, including obesity. The AT plasticity is strictly related to tissue vascularization. Angiogenesis supports the AT expansion, while regression of blood vessels is associated with AT hypoxia, which in turn mediates tissue inflammation, fibrosis and metabolic dysfunction. Several adipokines can regulate endothelial cell functions and are endowed with either pro- or anti-angiogenic properties. Here we address the role of adipokines in the regulation of angiogenesis. A better understanding of the link between adipokines and angiogenesis will open the way for novel therapeutic approaches to treat obesity and metabolic diseases.

Breeding in the pandemic: short-term lockdown restrictions in a European capital city did not alter the life-history traits of two urban adapters.

Humans are transforming natural habitats into managed urban green areas and impervious surfaces at an unprecedented pace. Yet the effects of human presence per se on animal life-history traits are rarely tested. This is particularly true in cities, where human presence is often indissociable from urbanisation itself. The onset of the SARS-CoV-2 outbreak, along with the resulting lockdown restrictions, offered a unique, "natural experiment" to investigate wildlife responses to a sudden reduction in human activity. We analysed four years of avian breeding data collected in a European capital city to test whether lockdown measures altered nestbox occupancy and life-history traits in terms of egg laying date, incubation duration and clutch size in two urban adapters: great tits (Parus major) and blue tits (Cyanistes caeruleus). Lockdown measures, which modulated human presence, did not influence any of the life-history traits investigated. In contrast, the interaction between year and tree cover, a distinct ecological attribute of the urban space, was positively associated with clutch size, a key avian life-history and reproductive trait. This highlights the importance of inter-year variation and habitat quality over human activity on urban wildlife reproduction. We discuss our results in the light of other urban wildlife studies carried out during the pandemic, inviting the scientific community to carefully interpret all lockdown-associated shifts in biological traits. Supplementary Information: The online version contains supplementary material available at 10.1007/s11252-022-01309-5.

Coping with novelty across an urban mosaic: Provisioning latency increases closer to roads and is associated with species-specific reproductive success in two urban adapters.

Most research on urban avian ecology has focused on population- and community-level phenomena, whereas fewer studies have examined how urbanization affects individual behavioral responses to a sudden and novel stimulus, and how those translate to fitness. We measured between-individual variation in provisioning latency in two urban adapters - great tits and blue tits - in response to an infrared camera installed in the nestbox, encountered when offspring in the nest were at the peak of food demand (9-10-days old). For each nestbox, we quantified urbanization as intensity in human activity, distance to road and proportion of impervious surface area. In both species, provisioning latency increased closer to roads. Moreover, increased provisioning latency when exposed to a novel object was associated with higher reproductive success in great tits whose nestboxes were surrounded by high amounts of impervious surface. In contrast, increased provisioning latency was consistently associated with lower reproductive success in blue tits. Our results suggest that provisioning latency changes in relation to the environment surrounding the nest, and may be context- and species-specific when exposed to a novel stimulus, such as a novel object in the nest. To better understand the role of initial behavioral responses towards novelty across an individual's lifetime and, ultimately, its impact on fitness in the urban mosaic, further research explicitly testing different behavioral responses across the entire breeding cycle in wild model systems is needed.

The extended avian urban phenotype: anthropogenic solid waste pollution, nest design, and fitness.

Solid waste pollution (garbage discarded by humans, such as plastic, metal, paper) has received increased attention given its importance as a global threat to biodiversity. Recent studies highlight how animals incorporate anthropogenic materials into their life-cycle, for example in avian nest construction. While increasingly monitored in natural areas, the influence of solid waste pollution on wildlife has been seldom explored in the urban habitat. There is limited data on the relationship between anthropogenic solid waste pollution, nest design, and reproductive success in an urban context. We address this knowledge gap (i) by investigating the presence of environmental solid waste pollution in the breeding habitats of great tits Parus major and blue tits Cyanistes caeruleus reproducing in a gradient of urbanisation, and (ii) by quantifying (ii) the contribution of different anthropogenic materials in their nests. We further examine potential drivers of solid waste pollution by inferring three distinct properties of the urban space: environmental solid waste pollution on the ground, human presence, and the intensity of urbanisation (e.g impervious surfaces) in nestbox vicinity. Finally, (iii) we explore the relationship between anthropogenic nest materials and reproductive success. We found that environmental solid waste pollution was positively associated with human presence and urbanisation intensity. There was also a positive relationship between increased human presence and the amount of anthropogenic materials in great tit nests. Interestingly, in both species, anthropogenic nest materials covaried negatively with nest materials of animal origin (fur and feathers). We suggest that fur and feathers - key insulating materials in nest design - may be scarcer in areas with high levels of human presence, and are consequently replaced with anthropogenic nest materials. Finally, we report a negative relationship between anthropogenic nest materials and blue tit reproductive success, suggesting species-specific vulnerability of urban birds to solid waste pollution.

Changes to the gut microbiota of a wild juvenile passerine in a multidimensional urban mosaic.

Urbanisation is a major anthropogenic perturbation presenting novel ecological and evolutionary challenges to wild populations. Symbiotic microorganisms residing in the gastrointestinal tracts (gut) of vertebrates have mutual connections with host physiology and respond quickly to environmental alterations. However, the impact of anthropogenic changes and urbanisation on the gut microbiota remains poorly understood, especially in early development. To address this knowledge gap, we characterised the gut microbiota of juvenile great tits (Parus major) reared in artificial nestboxes and in natural cavities in an urban mosaic, employing two distinct frameworks characterising the urban space. Microbial diversity was influenced by cavity type. Alpha diversity was affected by the amount of impervious surface surrounding the breeding location, and positively correlated with tree cover density. Community composition differed between urban and rural sites: these alterations covaried with sound pollution and distance to the city centre. Overall, the microbial communities reflect and are possibly influenced by the heterogeneous environmental modifications that are typical of the urban space. Strikingly, the choice of framework and environmental variables characterising the urban space can influence the outcomes of such ecological studies. Our results open new perspectives to investigate the impact of microbial symbionts on the adaptive capacity of their hosts.

Production and Biochemical Characterization of Dimeric Recombinant Gremlin-1.

Gremlin-1 is a secreted cystine-knot protein that acts as an antagonist of bone morphogenetic proteins (BMPs), and as a ligand of heparin and the vascular endothelial growth factor receptor 2 (VEGFR2), thus regulating several physiological and pathological processes, including embryonic development, tissue fibrosis and cancer. Gremlin-1 exerts all these biological activities only in its homodimeric form. Here, we propose a multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells. Ion metal affinity chromatography (IMAC) of crude supernatant followed by heparin-affinity chromatography enables obtaining a highly pure recombinant dimeric gremlin-1 protein, exhibiting both BMP antagonist and potent VEGFR2 agonist activities.

Irisin regulates thermogenesis and lipolysis in 3T3-L1 adipocytes.

BACKGROUND: Adipose tissue plays a pivotal role in the development and progression of the metabolic syndrome which along with its complications is an epidemic of the 21st century. Irisin is an adipo-myokine secreted mainly by skeletal muscle and targeting, among others, adipose tissue. In brown adipose tissue it upregulates uncoupling protein-1 (UCP1) which is responsible for mitochondrial non-shivering thermogenesis. METHODS: Here we analyzed the effects of irisin on the metabolic activity of 3T3-L1 derived adipocytes through a mitochondrial flux assay. We also assessed the effects of irisin on the intracellular signaling through Western Blot. Finally, the gene expression of ucp1 and lipolytic genes was examined through RT-qPCR. RESULTS: Irisin affects mitochondrial respiration and lipolysis in a time-dependent manner through the regulation of PI3K-AKT pathway. Irisin also induces the expression of UCP1 and the regulation of NF-κB, and CREB and ERK pathways. CONCLUSION: Our data supports the role of irisin in the induction of non-shivering thermogenesis, the regulation of energy expenditure and lipolysis in adipocytes. GENERAL SIGNIFICANCE: Irisin may be an attractive therapeutic target in the treatment of obesity and related metabolic disorders.

Novel potential oncogenic and druggable mutations of FGFRs recur in the kinase domain across cancer types.

Fibroblast growth factor receptors (FGFRs) are recurrently altered by single nucleotide variants (SNVs) in many human cancers. The prevalence of SNVs in FGFRs depends on the cancer type. In some tumors, such as the urothelial carcinoma, mutations of FGFRs occur at very high frequency (up to 60%). Many characterized mutations occur in the extracellular or transmembrane domains, while fewer known mutations are found in the kinase domain. In this study, we performed a bioinformatics analysis to identify novel putative cancer driver or therapeutically actionable mutations of the kinase domain of FGFRs. To pinpoint those mutations that may be clinically relevant, we exploited the recurrence of alterations on analogous amino acid residues within the kinase domain (PK_Tyr_Ser-Thr) of different kinases as a predictor of functional impact. By exploiting MutationAligner and LowMACA bioinformatics resources, we highlighted novel uncharacterized mutations of FGFRs which recur in other protein kinases. By revealing unanticipated correspondence with known variants, we were able to infer their functional effects, as alterations clustering on similar residues in analogous proteins have a high probability to elicit similar effects. As FGFRs represent an important class of oncogenes and drug targets, our study opens the way for further studies to validate their driver and/or actionable nature and, in the long term, for a more efficacious application of precision oncology.

Replicated, urban-driven exposure to metallic trace elements in two passerines.

While there are increasing examples of phenotypic and genotypic differences between urban and non-urban populations of plants and animals, few studies identified the mechanisms explaining those dissimilarities. The characterization of the urban landscape, which can only be achieved by measuring variability in relevant environmental factors within and between cities, is a keystone prerequisite to understand the effects of urbanization on wildlife. Here, we measured variation in bird exposure to metal pollution within 8 replicated urbanization gradients and within 2 flagship bird species in urban evolutionary ecology: the blue tit (Cyanistes caeruleus) and the great tit (Parus major). We report on a highly significant, positive linear relationship between the magnitude of urbanization-inferred as either tree cover, impervious surface cover, or an urbanization score computed from several environmental variables, and copper, zinc and lead concentrations in bird feathers. The reverse relationship was measured in the case of mercury, while cadmium and arsenic did not vary in response to the urbanization level. This result, replicated across multiple cities and two passerine species, strongly suggests that copper, zinc, lead and mercury pollution is likely to trigger the emergence of parallel responses at the phenotypic and/or genotypic level between urban environments worldwide.

Protein domain-based approaches for the identification and prioritization of therapeutically actionable cancer variants.

The tremendous number of cancer variants that can be detected by NGS analyses has required the development of computational approaches to prioritize mutations on the basis of their biological and clinical significance. Standard strategies take a gene-centric approach to the problem, allowing exclusively the identification of highly frequent variants. On the contrary, protein domain (PD)-based approaches allow to identify functionally relevant low frequency variants by searching for mutations that recur on analogous residues across homologous proteins (i.e. containing the same PD). Such approaches enable to transfer information about the effects and druggability from one known mutation to unknown ones. Here we describe how PD-based strategies work, and discuss how they could be exploited for mutation prioritization. The principle that mutations clustered on specific residues of PDs have the same functional consequences and are therapeutically actionable in a similar manner could help the choice of patient-specific targeted drugs, eventually improving the management of cancer patients.

Pentraxin 3 Inhibits the Angiogenic Potential of Multiple Myeloma Cells.

During multiple myeloma (MM) progression the activation of the angiogenic process represents a key step for the formation of the vascular niche, where different stromal components and neoplastic cells collaborate and foster tumor growth. Among the different pro-angiogenic players, Fibroblast Growth Factor 2 (FGF2) plays a pivotal role in BM vascularization occurring during MM progression. Long Pentraxin 3 (PTX3), a natural FGF antagonist, is able to reduce the activation of stromal components promoted by FGF2 in various in vitro models. An increased FGF/PTX3 ratio has also been found to occur during MM evolution, suggesting that restoring the "physiological" FGF/PTX3 ratio in plasma cells and BM stromal cells (BMSCs) might impact MM. In this work, taking advantage of PTX3-inducible human MM models, we show that PTX3 produced by tumor cells is able to restore a balanced FGF/PTX3 ratio sufficient to prevent the activation of the FGF/FGFR system in endothelial cells and to reduce the angiogenic capacity of MM cells in different in vivo models. As a result of this anti-angiogenic activity, PTX3 overexpression causes a significant reduction of the tumor burden in both subcutaneously grafted and systemic MM models. These data pave the way for the exploitation of PTX3-derived anti-angiogenic approaches in MM.

Expansion of tumor-associated Treg cells upon disruption of a CTLA-4-dependent feedback loop.

Foxp3+ T regulatory (Treg) cells promote immunological tumor tolerance, but how their immune-suppressive function is regulated in the tumor microenvironment (TME) remains unknown. Here, we used intravital microscopy to characterize the cellular interactions that provide tumor-infiltrating Treg cells with critical activation signals. We found that the polyclonal Treg cell repertoire is pre-enriched to recognize antigens presented by tumor-associated conventional dendritic cells (cDCs). Unstable cDC contacts sufficed to sustain Treg cell function, whereas T helper cells were activated during stable interactions. Contact instability resulted from CTLA-4-dependent downregulation of co-stimulatory B7-family proteins on cDCs, mediated by Treg cells themselves. CTLA-4-blockade triggered CD28-dependent Treg cell hyper-proliferation in the TME, and concomitant Treg cell inactivation was required to achieve tumor rejection. Therefore, Treg cells self-regulate through a CTLA-4- and CD28-dependent feedback loop that adjusts their population size to the amount of local co-stimulation. Its disruption through CTLA-4-blockade may off-set therapeutic benefits in cancer patients.

Expression of activated VEGFR2 by R1051Q mutation alters the energy metabolism of Sk-Mel-31 melanoma cells by increasing glutamine dependence.

Vascular endothelial growth factor receptor 2 (VEGFR2) activating mutations are emerging as important oncogenic driver events. Understanding the biological implications of such mutations may help to pinpoint novel therapeutic targets. Here we show that activated VEGFR2 via the pro-oncogenic R1051Q mutation induces relevant metabolic changes in melanoma cells. The expression of VEGFR2R1051Q leads to higher energy metabolism and ATP production compared to control cells expressing VEGFR2WT. Furthermore, activated VEGFR2R1051Q augments the dependence on glutamine (Gln) of melanoma cells, thus increasing Gln uptake and their sensitivity to Gln deprivation and to inhibitors of glutaminase, the enzyme initiating Gln metabolism by cells. Overall, these results highlight Gln addiction as a metabolic vulnerability of tumors harboring the activating VEGFR2R1051Q mutation and suggest novel therapeutic approaches for those patients harboring activating mutations of VEGFR2.

The Claudin-Low Subtype of High-Grade Serous Ovarian Carcinoma Exhibits Stem Cell Features.

Claudin-low cancer (CL) represents a rare and biologically aggressive variant of epithelial tumor. Here, we identified a claudin-low molecular profile of ovarian high-grade serous carcinoma (HGSOC), which exhibits the main characteristics of the homonym breast cancer subtype, including low epithelial differentiation and high mesenchymal signature. Hierarchical clustering and a centroid based algorithm applied to cell line collection expression dataset labeled 6 HGSOC cell lines as CL. These have a high energy metabolism and are enriched in CD44+/CD24- mesenchymal stem-like cells expressing low levels of cell-cell adhesion molecules (claudins and E-Cadherin) and high levels of epithelial-to-mesenchymal transition (EMT) induction transcription factors (Zeb1, Snai2, Twist1 and Twist2). Accordingly, the centroid base algorithm applied to large retrospective collections of primary HGSOC samples reveals a tumor subgroup with transcriptional features consistent with the CL profile, and reaffirms EMT as the dominant biological pathway functioning in CL-HGSOC. HGSOC patients carrying CL profiles have a worse overall survival when compared to others, likely to be attributed to its undifferentiated/stem component. These observations highlight the lack of a molecular diagnostic in the management of HGSOC and suggest a potential prognostic utility of this molecular subtyping.

Growing in the city: Urban evolutionary ecology of avian growth rates.

INTRODUCTION: Rapid environmental change driven by urbanization offers a unique insight into the adaptive potential of urban-dwelling organisms. Urban-driven phenotypic differentiation is increasingly often demonstrated, but the impact of urbanization (here modelled as the percentage of impervious surface (ISA) around each nestbox) on offspring developmental rates and subsequent survival remains poorly understood. Furthermore, the role of selection on urban-driven phenotypic divergence was rarely investigated to date. METHODS AND RESULTS: Data on nestling development and body mass were analysed in a gradient of urbanization set in Warsaw, Poland, in two passerine species: great tits (Parus major) and blue tits (Cyanistes caeruleus). Increasing levels of impervious surface area (ISA) delayed the age of fastest growth in blue tits. Nestling body mass was also negatively affected by increasing ISA 5 and 10 days after hatching in great tits, and 10 and 15 days in blue tits, respectively. High levels of ISA also increased nestling mortality 5 and 10 days after hatching in both species. An analysis of selection differentials performed for two levels of urbanization (low and high ISA) revealed a positive association between mass at day 2 and survival at fledging. DISCUSSION: This study confirms the considerable negative impact of imperviousness-a proxy for urbanization level-on offspring development, body mass and survival, and highlights increased selection on avian mass at hatching in a high ISA environment.

Simultaneously characterization of tumoral angiogenesis and vasculogenesis in stem cell-derived teratomas.

Tumor neovascularization may occur via both angiogenic and vasculogenic events. In order to investigate the vessel formation during tumor growth, we developed a novel experimental model that takes into account the differentiative and tumorigenic properties of Embryonic Stem cells (ESCs). Leukemia Inhibitory Factor-deprived murine ESCs were grafted on the top of the chick embryo chorionallantoic membrane (CAM) in ovo. Cell grafts progressively grew, forming a vascularized mass within 10 days. At this stage, the grafts are formed by cells with differentiative features representative of all three germ layers, thus originating teratomas, a germinal cell tumor. In addition, ESC supports neovascular events by recruiting host capillaries from surrounding tissue that infiltrates the tumor mass. Moreover, immunofluorescence studies demonstrate that perfused active blood vessels within the tumor are of both avian and murine origin because of the simultaneous occurrence of angiogenic and vasculogenic events. In conclusion, the chick embryo ESC/CAM-derived teratoma model may represent a useful approach to investigate both vasculogenic and angiogenic events during tumor growth and for the study of natural and synthetic modulators of the two processes.