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Under basal growth conditions, p53 function is tightly controlled by the members of MDM family, MDM2 and MDM4. The Mdm2 gene codes, in addition to the full-length p90MDM2, for a short protein, p76MDM2 that lacks the p53-binding domain. Despite this property and at variance with p90MDM2, this protein acts positively toward p53, although the molecular mechanism remains elusive. Here, we report that p76MDM2 antagonizes MDM4 inhibitory function. We show that p76MDM2 possesses intrinsic ubiquitinating and degrading activity, and through these activities controls MDM4 levels. Furthermore, the presence of p76MDM2 decreases the association of MDM4 with p53 and p90MDM2, and antagonizes p53 degradation by the heterodimer MDM4/p90MDM2. The p76MDM2-mediated regulation of MDM4 occurs in the cytoplasm, under basal growth conditions. Conversely, upon DNA damage, phosphorylation of MDM4Ser403 dissociates p76MDM2 and prevents MDM4 degradation. The overall negative control of MDM4 by p76MDM2 reflects on p53 function as p76MDM2 impairs MDM4-mediated inhibition of p53 activity. In agreement with the positive role of p76MDM2 toward p53, the p76MDM2/p90MDM2 ratio significantly decreases in a group of thyroid tumor samples compared with normal counterparts. Overall, these findings reveal a new mechanism in the control of p53 basal activity that may account for the distinct sensitivity of tissues to stress signals depending on the balance among MDM proteins. Moreover, these data suggest an oncosuppressive function for a product of the Mdm2 gene. [ABSTRACT FROM AUTHOR]

Primary or secondary (i.e., acquired) resistance is a common occurrence in cancer patients and is often associated with high numbers of T regulatory (T reg ) cells (CD4 + CD25 + FOXP3 + ). The approval of ipilimumab and the development of similar pharmacological agents targeting cell surface proteins on T reg cells demonstrates that such intervention may overcome resistance in cancer patients. Hence, the clinical development and subsequent approval of Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) targeting agents can serve as a prototype for similar agents. Such new agents aspire to be highly specific and have a reduced toxicity profile while increasing effector T cell function or effector T/T regulatory (T eff /T reg ) ratio. While clinical development with large molecules has shown the greatest advancement, small molecule inhibitors that target immunomodulation are increasingly entering early clinical investigation. These new small molecule inhibitors often target specific intracellular signaling pathways [e.g., phosphoinositide-3-kinase delta (PI3K-δ)] that play an important role in regulating the function of T reg cells. This review will summarize the lessons currently applied to develop novel clinical agents that target T reg cells., Competing Interests: Spiliopoulou P declared that there are no conflicts of interest. Kaur P, Hammett T, and Lahn M are full-time employees of iOnctura SA; Di Conza G and Lahn M holds stocks in iOnctura SA., (© The Author(s) 2024.)

The TGFβ receptor inhibitor galunisertib demonstrated efficacy in patients with pancreatic ductal adenocarcinoma (PDAC) in the randomized phase II H9H-MC-JBAJ study, which compared galunisertib plus the chemotherapeutic agent gemcitabine with gemcitabine alone. However, additional stromal paracrine signals might confer adaptive resistance that limits the efficacy of this therapeutic strategy. Here, we found that autotaxin, a secreted enzyme that promotes inflammation and fibrosis by generating lysophosphatidic acid (LPA), mediates adaptive resistance to TGFβ receptor inhibition. Blocking TGFβ signaling prompted the skewing of cancer-associated fibroblasts (CAF) toward an inflammatory (iCAF) phenotype. iCAFs were responsible for a significant secretion of autotaxin. Paracrine autotaxin increased LPA-NFκB signaling in tumor cells that triggered treatment resistance. The autotaxin inhibitor IOA-289 suppressed NFκB activation in PDAC cells and overcame resistance to galunisertib and gemcitabine. In immunocompetent orthotopic murine models, IOA-289 synergized with galunisertib in restoring sensitivity to gemcitabine. Most importantly, treatment with galunisertib significantly increased plasma levels of autotaxin in patients enrolled in the H9H-MC-JBAJ study, and median progression-free survival was significantly longer in patients without an increase of autotaxin upon treatment with galunisertib compared with those with increased autotaxin. These results establish that autotaxin secretion by CAFs is increased by TGFβ inhibition and that circulating autotaxin levels predict response to the combination treatment approach of gemcitabine plus galunisertib., Significance: TGFβ inhibition skews cancer-associated fibroblasts toward an inflammatory phenotype that secretes autotaxin to drive adaptive resistance in PDAC, revealing autotaxin as a therapeutic target and biomarker of galunisertib response., (©2023 The Authors; Published by the American Association for Cancer Research.)

We engineered an in vitro model of bioartificial 3D bone organoid consistent with an anatomical and vascular microenvironment common to mammalian flat and short bones. To achieve this, we chose the decellularized-decalcified matrix of the adult male rat scapula, implemented with the reconstruction of its intrinsic vessels, obtained through an original intravascular perfusion with polylevolactic (PLLA), followed by coating of the PLLA-fabricated vascularization with rat tail collagen. As a result, the 3D bone and vascular geometry of the native bone cortical and cancellous compartments was reproduced, and the rat tail collagen-PLLA biomaterial could in vitro act as a surrogate of the perivascular extracellular matrix (ECM) around the wall of the biomaterial-reconstituted cancellous vessels. As a proof-of-concept of cell compatibility and site-dependent osteoinductive properties of this bioartificial 3D construct, we show that it in vitro leads to a time-dependent microtopographic positioning of rat mesenchymal stromal cells (MSCs), initiating an osteogenic fate in relation to the bone compartment. In addition, coating of PLLA-reconstructed vessels with rat tail collagen favored perivascular attachment and survival of MSC-like cells (mouse embryonic fibroblasts), confirming its potentiality as a perivascular stroma for triggering competence of seeded MSCs. Finally, in vivo radiographic topography of bone lesions in the human jaw and foot tarsus of subjects with primary osteoporosis revealed selective bone cortical versus cancellous involvement, suggesting usefulness of a human 3D bone organoid engineered with the same principles of our rat organoid, to in vitro investigate compartment-dependent activities of human MSC in flat and short bones under experimental osteoporotic challenge. We conclude that our 3D bioartificial construct offers a reliable replica of flat and short bones microanatomy, and promises to help in building a compartment-dependent mechanistic perspective of bone remodeling, including the microtopographic dysregulation of osteoporosis., (© 2023 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals LLC.)

Osteoporosis and fragility fractures (FFs) are considered critical health problems by the World Health Organization (WHO) because of high morbidity, mortality, and healthcare costs. The occurrence of a FF raises the risk of a subsequent fracture (refracture). The hip is the most common site of fragility refracture, and its onset is associated with a further increase in patient's morbidity, mortality, and socioeconomic burden. Therefore, the prevention of refracture is essential. In this context, fracture liaison service (FLS) demonstrated to be able to reduce FF risk and also improve patients' adherence to anti-osteoporotic treatments, particularly for bisphosphonates (BPs). However, long-term and high adherence to BPs may lead to atypical femoral fractures (AFFs). These latter are tensile side stress fractures of the femur, with high rates of complications, including delayed and non-healing. An effective FLS should be able to prevent both FF and AFF. A comprehensive and interdisciplinary approach, through the involvement and education of a dedicated team of healthcare professionals (i.e. orthopedic, geriatrician, primary care physician, rehabilitation team, and bone nurse) for evaluating both FF and AFF risks might be useful to improve the standard of care., Competing Interests: UT serves on the Advisory Board for Abiogen, AMGEN, and Eli-Lilly. GI serves on the Advisory Board for UCB Pharma, AMGEN, and Eli-Lilly. The other authors declare that there is no conflict of interest., (© The Author(s), 2023.)

Background: Disordered and hypomineralized woven bone formation by dysfunctional mesenchymal stromal cells (MSCs) characterize delayed fracture healing and endocrine -metabolic bone disorders like fibrous dysplasia and Paget disease of bone. To shed light on molecular players in osteoblast differentiation, woven bone formation, and mineralization by MSCs we looked at the intermediate filament desmin (DES) during the skeletogenic commitment of rat bone marrow MSCs (rBMSCs), where its bone-related action remains elusive., Results: Monolayer cultures of immunophenotypically- and morphologically - characterized, adult male rBMSCs showed co-localization of desmin (DES) with vimentin, F-actin, and runx2 in all cell morphotypes, each contributing to sparse and dense colonies. Proteomic analysis of these cells revealed a topologically-relevant interactome, focused on cytoskeletal and related enzymes//chaperone/signalling molecules linking DES to runx2 and alkaline phosphatase (ALP). Osteogenic differentiation led to mineralized woven bone nodules confined to dense colonies, significantly smaller and more circular with respect to controls. It significantly increased also colony-forming efficiency and the number of DES-immunoreactive dense colonies, and immunostaining of co-localized DES/runx-2 and DES/ALP. These data confirmed pre-osteoblastic and osteoblastic differentiation, woven bone formation, and mineralization, supporting DES as a player in the molecular pathway leading to the osteogenic fate of rBMSCs., Conclusion: Immunocytochemical and morphometric studies coupled with proteomic and bioinformatic analysis support the concept that DES may act as an upstream signal for the skeletogenic commitment of rBMSCs. Thus, we suggest that altered metabolism of osteoblasts, woven bone, and mineralization by dysfunctional BMSCs might early be revealed by changes in DES expression//levels. Non-union fractures and endocrine - metabolic bone disorders like fibrous dysplasia and Paget disease of bone might take advantage of this molecular evidence for their early diagnosis and follow-up., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Di Conza, Barbaro, Zini, Spaletta, Remaggi, Elviri, Mosca, Caravelli, Mosca and Toni.)

Initiating and maintaining optimal immune responses requires high levels of protein synthesis, folding, modification and trafficking in leukocytes, which are processes orchestrated by the endoplasmic reticulum. Importantly, diverse extracellular and intracellular conditions can compromise the protein-handling capacity of this organelle, inducing a state of 'endoplasmic reticulum stress' that activates the unfolded protein response (UPR). Emerging evidence shows that physiological or pathological activation of the UPR can have effects on immune cell survival, metabolism, function and fate. In this Review, we discuss the canonical role of the adaptive UPR in immune cells and how dysregulation of this pathway in leukocytes contributes to diverse pathologies such as cancer, autoimmunity and metabolic disorders. Furthermore, we provide an overview as to how pharmacological approaches that modulate the UPR could be harnessed to control or activate immune cell function in disease., (© 2023. Springer Nature Limited.)

Strategies to increase intratumoral concentrations of an anticancer agent are desirable to optimize its therapeutic potential when said agent is efficacious primarily within a tumor but also have significant systemic side effects. Here, we generate a bifunctional protein by fusing interleukin-10 (IL-10) to a colony-stimulating factor-1 receptor (CSF-1R)-blocking antibody. The fusion protein demonstrates significant antitumor activity in multiple cancer models, especially head and neck cancer. Moreover, this bifunctional protein not only leads to the anticipated reduction in tumor-associated macrophages but also triggers proliferation, activation, and metabolic reprogramming of CD8 + T cells. Furthermore, it extends the clonotype diversity of tumor-infiltrated T cells and shifts the tumor microenvironment (TME) to an immune-active state. This study suggests an efficient strategy for designing immunotherapeutic agents by fusing a potent immunostimulatory molecule to an antibody targeting TME-enriched factors., Competing Interests: Declaration of interests P.-C.H. is a scientific advisor for Elixiron Immunotherapeutics, Acepodia, and Novartis and is a cofounder of Pilatus Biosciences. P.-C.H. received research support from Elixiron Immunotherapeutics. P.-C.H. also received research support from Roche., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Autotaxin (ATX) is a secreted enzyme that converts lysophosphatidylcholine to lysophosphatidic acid (LPA). LPA stimulates cell proliferation and migration and promotes wound repair following tissue damage. ATX levels are directly correlated with stage and grade in several human cancers. Several small molecule ATX inhibitors have been developed in recent years. IOA-289 is a potent ATX inhibitor, developed to treat cancers containing fibrosis. In this study, we tested IOA-289 treatment on different gastrointestinal tract tumor cell lines, in order to evaluate its effects on viability and motility., Methods: To determine the effects on cell viability and proliferation of treatment with increasing concentrations of IOA-289, we used the crystal violet assay, a clonogenic assay in matrigel, and we evaluated the inhibitor's effect on formation of 3D spheroids in an in vitro model. The effect of IOA-289 on cell cycle phases was analysed with a redox dye reagent. Cell migration capacity was evaluated by wound healing assay and transwell migration assay. To evaluate the pro-apoptotic effect of the inhibitor, cells were stained with Annexin V and immunofluorescence and flow cytometry analysis were performed. An antibody array was also used, to discriminate, in various samples, the differential expression of 43 proteins involved in the apoptosis pathway., Results: We found that IOA-289 is able to inhibit both growth and migration of gastrointestinal tract tumor cell lines, both in 2D (crystal violet assay) and 3D in vitro models (spheroid formation and clonogenic assay in matrigel). This effect is dose-dependent, and the drug is most effective when administered in FBS-free culture medium. The inhibitory effect on cell growth is due to a pro-apoptotic effect of IOA-289. Staining with FITC-conjugated Annexin V showed that IOA-289 induced a dose-dependent increase in fluorescence following incubation for 24 h, and apoptotic cells were also distinguished in flow cytometry using Annexin/PI staining. The antibody array shows that treatment with IOA-289 causes the increased expression of several pro-apoptotic proteins in all tested cell lines., Conclusions: These results indicate that IOA-289 may be an effective drug for the treatment of tumors of the gastrointestinal tract, particularly those characterized by a high degree of fibrosis., (© 2023. Italian National Cancer Institute ‘Regina Elena’.)

PI3K delta (PI3Kδ) inhibitors are used to treat lymphomas but safety concerns and limited target selectivity curbed their clinical usefulness. PI3Kδ inhibition in solid tumors has recently emerged as a potential novel anticancer therapy through the modulation of T-cell responses and direct antitumor activity. Here we report the exploration of IOA-244/MSC2360844, a first-in-class non-ATP-competitive PI3Kδ inhibitor, for the treatment of solid tumors. We confirm IOA-244's selectivity as tested against a large set of kinases, enzymes, and receptors. IOA-244 inhibits the in vitro growth of lymphoma cells and its activity correlates with the expression levels of PIK3CD , suggesting cancer cell-intrinsic effects of IOA-244. Importantly, IOA-244 inhibits regulatory T cell proliferation while having limited antiproliferative effects on conventional CD4 + T cells and no effect on CD8 + T cells. Instead, treatment of CD8 T cells with IOA-244 during activation, favors the differentiation of memory-like, long-lived CD8, known to have increased antitumor capacity. These data highlight immune-modulatory properties that can be exploited in solid tumors. In CT26 colorectal and Lewis lung carcinoma lung cancer models, IOA-244 sensitized the tumors to anti-PD-1 (programmed cell death protein 1) treatment, with similar activity in the Pan-02 pancreatic and A20 lymphoma syngeneic mouse models. IOA-244 reshaped the balance of tumor-infiltrating cells, favoring infiltration of CD8 and natural killer cells, while decreasing suppressive immune cells. IOA-244 presented no detectable safety concerns in animal studies and is currently in clinical phase Ib/II investigation in solid and hematologic tumors., Significance: IOA-244 is a first-in-class non-ATP-competitive, PI3Kδ inhibitor with direct antitumor in vitro activity correlated with PI3Kδ expression. The ability to modulate T cells, in vivo antitumor activity in various models with limited toxicity in animal studies provides the rationale for the ongoing trials in patients with solid tumors and hematologic cancers., (© 2023 The Authors; Published by the American Association for Cancer Research.)

We recently designed an innovative scaffold-bioreactor unit for the bioengineering of a three-dimensional (3D) bioartificial human thyroid gland or its miniaturized replica as a part of a microfluidic chip test system. This device is based on the evidence that the 3D geometry of the intraglandular stromal/vascular scaffold (SVS; i.e., the fibrous and vascular matrix) of mammalian viscera plays a key role in guiding growth and differentiation of in vitro seeded cells. Therefore, we initiated a research program focused on computer-aided reconstruction of the 2nd to 4th order intralobar arterial network (IAN) of the human thyroid gland as a reliable surrogate for its 3D SVS, to be used as an input for rapid prototyping of a biomaterial replica. To this end, we developed a computational template that works within the Mathematica environment, giving rise to a quasi-fractal growth of the IAN distribution, constrained within an approximation of the thyroid lobe shape as a closed surface. Starting from edge detection of planar images of real human thyroid lobes acquired by in vivo real-time ultrasonography, we performed data approximation of the lobar profiles based on splines and Bezier curves, providing 3D lobar shapes as geometric boundaries for vessel growth by a diffusion-limited aggregation model. Our numerical procedures allowed for a robust connection between development of lobar arterial trees and thyroid lobe shape, led to a vascular self-similarity consistent with that of a cadaveric lobar arterial cast, and reproduced arterial vessels in a proportion not statistically different from that described for the real human thyroid gland. We conclude that our algorithmic template offers a reliable reproduction of the extremely complex IAN of the adult human thyroid lobe, potentially useful as a computational guidance for bioprinting of thyroid lobe matrix replicas. In addition, due to the simplicity and limited number of morphometrical parameters required by our system, we predict its application to the design of a number of patient-tailored human bioartificial organs and organs-on-chip, including parenchymal viscera and bones. Impact statement The study introduces the computer simulation of the three-dimensional (3D) intrinsic vascular matrix of the human thyroid gland, offering a general concept applicable to a number of other human viscera. Indeed, it provides a flexible software tool for reproduction of a 3D surrogate of the organ's 3D stromal matrix, suitable for eventual 3D bioprinting with biomaterials, and recellularization with organ-specific stem cells/progenitors. The final expectation is the design of patient-tailored 3D organ's matrices upon clinical request.

T cells dynamically rewire their metabolism during an immune response. We applied single-cell RNA sequencing to CD8 + T cells activated and differentiated in vitro in physiological medium to resolve these metabolic dynamics. We identify a differential time-dependent reliance of activating T cells on the synthesis versus uptake of various non-essential amino acids, which we corroborate with functional assays. We also identify metabolic genes that potentially dictate the outcome of T cell differentiation, by ranking them based on their expression dynamics. Among them, we find asparagine synthetase (Asns), whose expression peaks for effector T cells and decays toward memory formation. Disrupting these expression dynamics by ASNS overexpression promotes an effector phenotype, enhancing the anti-tumor response of adoptively transferred CD8 + T cells in a mouse melanoma model. We thus provide a resource of dynamic expression changes during CD8 + T cell activation and differentiation, and identify ASNS expression dynamics as a modulator of CD8 + T cell differentiation., Competing Interests: Declaration of interests P.-C.H. is on the scientific advisory board for Elixiron Immunotherapeutics, Acepodia, and Novartis; has received funding from Elixiron Immunotherapeutics; and is the founder of Pilatus Biosciences. S.-M.F. has received funding from Bayer, Merck, Black Belt Therapeutics, and Alesta Therapeutics; has consulted for Fund+; and is on the scientific advisory board for Alesta Therapeutics and the editorial board of Cell Reports., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: IM nails are the gold standard of subtrochanteric fractures management. Indications to use a short rather than a long nail remain unclear. Operative complications of subtrochanteric fractures reach up to 25%., Objective: Retrospectively compare clinical and radiographic outcome of subtrochanteric fractures treated by long and short intramedullary nailing, analysing rates of complications., Methods: 390 patients were chosen from the archives. 194 patients were available: 70 treated with a short intramedullary nail (Group A), while 124 with long one (Group B). Radiographic evaluation at 6 and 12 months assess failure of the osteosynthesis. Clinical outcomes were the return to normal activities prior trauma and VAS scale. Group A mean age was 81.37 years, group B mean age was 78.9 years (29-99, SD: 15.38)., Results: Radiografic Healing was found in 66 patients (94.28%) in group A, while in 116 patients (94.54%) in group B. Pseudarthrosis was found in 4 cases (5.71%) in group A, while in 8 cases (6.45%) in group B. Implant failure occurred in 5 cases: one required revision of fixation, while 4 require hip replacement. 59 patients of group A (84.29%) returned to social life, while 102 patients (81.94%) in group B. Group A mean VAS was 1.55, Group B mean VAS was 1.49., Conclusion: Comparison of the two group showed no differences. Complication percentages are in line with literature. Optimal reduction and fixation allow high percentage of healing and return to social life., Competing Interests: The authors declare that they have no conflict of interest

Macrophages are main players of the innate immune system. They show great heterogeneity and play diverse functions that include support to development, sustenance of tissue homeostasis and defense against infections. Dysfunctional macrophages have been described in multiple pathologies including cancer. Indeed tumor-associated macrophages (TAMs) are abundant in most tumors and sustain cancer growth, promote invasion and mediate immune evasion. Importantly, lipid metabolism influences macrophage activation and lipid accumulation confers pathogenic features on macrophages. Notably, a subset of lipid-loaded macrophages has been recently identified in many tumor types. Lipid-loaded TAMs support tumor growth and progression and exert immune-suppressive activities. In this review, we describe the role of lipid metabolism in macrophage activation in physiology and pathology and we discuss the impact of lipid accumulation in macrophages in the context of cancer., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Collagens, elastin, fibrillin, decorin, and laminin are key constituents of the extracellular matrix and basement membrane of mammalian organs. Thus, changes in their quantities may influence the mechanochemical regulation of resident cells. Since maintenance of a native stromal composition is a requirement for three-dimensional (3D) matrix-based recellularization techniques in tissue engineering, we studied the influence of the decellularization detergents on these proteins in porcine kidney, liver, pancreas, and skin. Using a quick thawing/quick microwave-assisted decellularization protocol and two different detergents, sodium dodecyl sulfate (SDS) vs Triton X-100 (TX100), at identical concentration, variations in matrix conservation of stromal proteins were detected by liquid chromatography-mass spectrometry coupled to light and scanning electron microscopies, in dependence on each detergent. In all organs tested except pancreas, collagens were retained to a statistically significant level using the TX100-based protocol. In contrast fibrillin, elastin (except in kidney), and decorin (only in liver) were better preserved with the SDS-dependent protocol. Irrespective of the detergent used, laminin always remained at an irrelevant level. Our results prompt attention to the type of detergent in organ decellularization, suggesting that its choice may influence morphoregulatory inputs peculiar to the type of 3D bioartificial mammalian organ to be reconstructed. Impact statement Simple change of the protocol's main detergent leads to a very substantial difference in the panel of the stromal proteins detected by qualitative and semiquantitative mass spectrometry in acellular porcine matrices. This remarkable methodological variable promises to yield proteomic reference panels in a number of different species-specific acellular matrices allowing for selective retainment of peculiar mechanochemical inputs, to differently address the development of the seeded cells in relation to the type of organ to be bioartificially reconstructed.

Tumor-associated macrophages (TAMs) display pro-tumorigenic phenotypes for supporting tumor progression in response to microenvironmental cues imposed by tumor and stromal cells. However, the underlying mechanisms by which tumor cells instruct TAM behavior remain elusive. Here, we uncover that tumor-cell-derived glucosylceramide stimulated unconventional endoplasmic reticulum (ER) stress responses by inducing reshuffling of lipid composition and saturation on the ER membrane in macrophages, which induced IRE1-mediated spliced XBP1 production and STAT3 activation. The cooperation of spliced XBP1 and STAT3 reinforced the pro-tumorigenic phenotype and expression of immunosuppressive genes. Ablation of XBP1 expression with genetic manipulation or ameliorating ER stress responses by facilitating LPCAT3-mediated incorporation of unsaturated lipids to the phosphatidylcholine hampered pro-tumorigenic phenotype and survival in TAMs. Together, we uncover the unexpected roles of tumor-cell-produced lipids that simultaneously orchestrate macrophage polarization and survival in tumors via induction of ER stress responses and reveal therapeutic targets for sustaining host antitumor immunity., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Osteoporosis stems from an unbalance between bone mineral resorption and deposition. Among the numerous cellular players responsible for this unbalance bone marrow (BM) monocytes/macrophages, mast cells, T and B lymphocytes, and dendritic cells play a key role in regulating osteoclasts, osteoblasts, and their progenitor cells through interactions occurring in the context of the different bone compartments (cancellous and cortical). Therefore, the microtopography of immune cells inside trabecular and compact bone is expected to play a relevant role in setting initial sites of osteoporotic lesion. Indeed, in physiological conditions, each immune cell type preferentially occupies either endosteal, subendosteal, central, and/or perisinusoidal regions of the BM. However, in the presence of an activation, immune cells recirculate throughout these different microanatomical areas giving rise to a specific distribution. As a result, the trabeculae of the cancellous bone and endosteal free edge of the diaphyseal case emerge as the primary anatomical targets of their osteoporotic action. Immune cells may also transit from the BM to the depth of the compact bone, thanks to the efferent venous capillaries coursing in the Haversian and Volkmann canals. Consistently, the innermost parts of the osteons and the periosteum are later involved by their immunomodulatory action, becoming another site of mineral reabsorption in the course of an osteoporotic insult. The novelty of our updating is to highlight the microtopography of bone immune cells in the cancellous and cortical compartments in relation to the most consistent data on their action in bone remodeling, to offer a mechanist perspective useful to dissect their role in the osteoporotic process, including bone damage derived from the immunomodulatory effects of endocrine disrupting chemicals., (Copyright © 2020 Toni, Di Conza, Barbaro, Zini, Consolini, Dallatana, Antoniel, Quarantini, Quarantini, Maioli, Bruni, Elviri, Panseri, Sprio, Sandri and Tampieri.)

Rationale: How endothelial cells (ECs) migrate and form an immature vascular plexus has been extensively studied. Yet, mechanisms underlying vascular remodeling remain poorly established. A better understanding of these processes may lead to the design of novel therapeutic strategies complementary to current angiogenesis inhibitors., Objective: Starting from our previous observations that PP2A (protein phosphatase 2) regulates the HIF (hypoxia-inducible factor)/PHD-2 (prolyl hydroxylase 2)-constituted oxygen machinery, we hypothesized that this axis could play an important role during blood vessel formation, tissue perfusion, and oxygen restoration., Methods and Results: We show that the PP2A regulatory subunit B55α is at the crossroad between vessel pruning and vessel maturation. Blood vessels with high B55α counter cell stress conditions and thrive for stabilization and maturation. When B55α is inhibited, ECs cannot cope with cell stress and undergo apoptosis, leading to massive pruning of nascent blood vessels. Mechanistically, we found that the B55α/PP2A complex restrains PHD-2 activity, promoting EC survival in a HIF-dependent manner, and furthermore dephosphorylates p38, altogether protecting ECs against cell stress occurring, for example, during the onset of blood flow. In tumors, EC-specific B55α deficiency induces pruning of immature-like tumor blood vessels resulting in delayed tumor growth and metastasis, without affecting nonpathological vessels. Consistently, systemic administration of a pan-PP2A inhibitor disrupts vascular network formation and tumor progression in vivo without additional effects on B55α-deficient vessels., Conclusions: Our data underline a unique role of the B55α/PP2A phosphatase complex in vessel remodeling and suggest the use of PP2A-inhibitors as potent antiangiogenic drugs targeting specifically nascent blood vessels with a mode-of-action complementary to VEGF-R (vascular endothelial growth factor receptor)-targeted therapies. Graphical Abstract: A graphical abstract is available for this article.

The endoplasmic reticulum (ER) is a critical organelle, storing the majority of calcium and governing protein translation. Thus, it is crucial to keep the homeostasis in all ER components and machineries. The ER stress sensor pathways, including IRE1/sXBP1, PERK/EIf2 and ATF6, orchestrate the major regulatory circuits to ensure ER homeostasis. The embryonic or postnatal lethality that occurs upon genetic depletion of these sensors reveals the essential role of the ER stress pathway in cell biology. In contrast, the impairment or excessive activation of ER stress has been reported to cause or aggravate several diseases such as atherosclerosis, diabetes, NAFDL/NASH, obesity and cancer. Being part of innate immunity, myeloid cells are the first immune cells entering the inflammation site. Upon entry into a metabolically stressed disease environment, activation of ER stress occurs within the myeloid compartment, leading to the modulation of their phenotype and functions. In this review, we discuss causes and consequences of ER stress activation in the myeloid compartment with a special focus on the crosstalk between ER, innate signaling and metabolic environments., Competing Interests: P.-C.H. is a member of scientific advisory board for Elixiron Immunotherapeutics and receiving research support from Idorsia and Roche.

An academic, anatomist, and Lombrosian psychiatrist active at the University of Parma in Italy at the end of the 19th century, Lorenzo Tenchini produced ceroplastic-like masks that are unique in the anatomical Western context. These were prepared from 1885 to 1893 with the aim of 'cataloguing' the behaviour of prison inmates and psychiatric patients based on their facial surface anatomy. Due to the lack of any reference to the procedure used to prepare the masks, studies were undertaken by our group using X-ray scans, infrared spectroscopy, bioptic sampling, and microscopy analysis of the mask constituents. Results showed that the masks were stratified structures including plaster, cotton gauze/human epidermis, and wax, leading to a fabrication procedure reminiscent of 'additive layer manufacturing'. Differences in the depths of these layers were observed in relation to the facial contours, suggesting an attempt to reproduce, at least partially, the three-dimensional features of the facial soft tissues. We conclude the Tenchini masks are the first historical antecedent of the experimental method for face reconstruction used in the early 2000s to test the feasibility of transferring a complete strip of face and scalp from a deceased donor to a living recipient, in preparation for a complete face transplant. In addition, the layering procedure adopted conceptually mimics that developed only in the late 20th century for computer-aided rapid prototyping, and recently applied to bioengineering with biomaterials for a number of human structures including parts of the skull and face. Finally, the masks are a relevant example of mixed ceroplastic-cutaneous preparations in the history of anatomical research for clinical purposes., (© 2019 Anatomical Society.)

High plasticity to utilize different nutrients to adapt metabolic stress is one of the hallmarks for cancer cells. However, the underlying mechanisms by which cancer cells reprogram metabolic machinery in response to metabolic stress remain largely unclear. In this issue of Molecular Cell, Wang et al. (2019) report that glutamate dehydrogenase 1 (GDH1) induces an unconventional regulation of the NF-κB pathway under glucose deprivation, thereby stimulating glycolysis in glioblastomas., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In response to physiological and pathological stimuli, macrophages are able to adapt and shape their phenotype, giving rise to a broad range of functional activation that is unique in different organs and different pathologies. The plasticity of macrophages is accomplished not only by di stinct signalling pathways and transcriptional profiles but also by specific engagement of preferential metabolic pathways. In the last decade, macrophage metabolism became the object of multiple studies showing that, by altering nutrient availability or by blocking specific metabolic pathway it is possible to skew macrophage phenotype and alter their effector functions. This field of research opens new therapeutic windows for the cure of several disease. Here we will give an overview of the current knowledge of macrophage metabolism in cancer, atherosclerosis and obesity and how this knowledge could be translated in therapeutic opportunities., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Although it is well established that tumor-associated macrophages take part in each step of cancer progression, less is known about the distinct role of the so-called metastasis-associated macrophages (MAMs) at the metastatic site. Previous studies reported that Caveolin-1 (Cav1) has both tumor-promoting and tumor-suppressive functions. However, the role of Cav1 in bone-marrow-derived cells is unknown. Here, we describe Cav1 as an anti-metastatic regulator in mouse models of lung and breast cancer pulmonary metastasis. Among all the recruited inflammatory cell populations, we show that MAMs uniquely express abundant levels of Cav1. Using clodronate depletion of macrophages, we demonstrate that macrophage Cav1 signaling is critical for metastasis and not for primary tumor growth. In particular, Cav1 inhibition does not affect MAM recruitment to the metastatic site but, in turn, favors angiogenesis. We describe a mechanism by which Cav1 in MAMs specifically restrains vascular endothelial growth factor A/vascular endothelial growth factor receptor 1 (VEGF-A/VEGFR1) signaling and its downstream effectors, matrix metallopeptidase 9 (MMP9) and colony-stimulating factor 1 (CSF1)., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Glutamine metabolism provides synergistic support for macrophage activation and elicitation of desirable immune responses; however, the underlying mechanisms regulated by glutamine metabolism to orchestrate macrophage activation remain unclear. Here we show that the production of α-ketoglutarate (αKG) via glutaminolysis is important for alternative (M2) activation of macrophages, including engagement of fatty acid oxidation (FAO) and Jmjd3-dependent epigenetic reprogramming of M2 genes. This M2-promoting mechanism is further modulated by a high αKG/succinate ratio, whereas a low ratio strengthens the proinflammatory phenotype in classically activated (M1) macrophages. As such, αKG contributes to endotoxin tolerance after M1 activation. This study reveals new mechanistic regulations by which glutamine metabolism tailors the immune responses of macrophages through metabolic and epigenetic reprogramming.

B55α is a regulatory subunit of the PP2A phosphatase. We have recently found that B55α-associated PP2A promotes partial deactivation of the HIF-prolyl-hydroxylase enzyme PHD2. Here, we show that, in turn, PHD2 triggers degradation of B55α by hydroxylating it at proline 319. In the context of glucose starvation, PHD2 reduces B55α protein levels, which correlates with MDA-MB231 and MCF7 breast cancer cell death. Under these conditions, PHD2 silencing rescues B55α degradation, overcoming apoptosis, whereas in SKBR3 breast cancer cells showing resistance to glucose starvation, B55α knockdown restores cell death and prevents neoplastic growth in vitro. Treatment of MDA-MB231-derived xenografts with the glucose competitor 2-deoxy-glucose leads to tumor regression in the presence of PHD2. Knockdown of PHD2 induces B55α accumulation and treatment resistance by preventing cell apoptosis. Overall, our data unravel B55α as a PHD2 substrate and highlight a role for PHD2-B55α in the response to nutrient deprivation., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Many tumor-related factors have shown the ability to affect metabolic pathways by paving the way for cancer-specific metabolic features. Here, we investigate the regulation of mTORC1 by MDM4, a p53-inhibitor with oncogenic or anti-survival activities depending on cell growth conditions., Method: MDM4-mTOR relationship was analysed through experiments of overexpression or silencing of endogenous proteins in cell culture and using purified proteins in vitro. Data were further confirmed in vivo using a transgenic mouse model overexpressing MDM4. Additionally, the Cancer Genome Atlas (TCGA) database (N = 356) was adopted to analyze the correlation between MDM4 and mTOR levels and 3D cultures were used to analyse the p53-independent activity of MDM4., Results: Following nutrient deprivation, MDM4 impairs mTORC1 activity by binding and inhibiting the kinase mTOR, and contributing to maintain the cytosolic inactive pool of mTORC1. This function is independent of p53. Inhibition of mTORC1 by MDM4 results in reduced phosphorylation of the mTOR downstream target p70S6K1 both in vitro and in vivo in a MDM4-transgenic mouse. Consistently, MDM4 reduces cell size and proliferation, two features controlled by p70S6K1, and, importantly, inhibits mTORC1-mediated mammosphere formation. Noteworthy, MDM4 transcript levels are significantly reduced in breast tumors characterized by high mTOR levels., Conclusion: Overall, these data identify MDM4 as a nutrient-sensor able to inhibit mTORC1 and highlight its metabolism-related tumor-suppressing function.

Oxygen-dependent HIF1α hydroxylation and degradation are strictly controlled by PHD2. In hypoxia, HIF1α partly escapes degradation because of low oxygen availability. Here, we show that PHD2 is phosphorylated on serine 125 (S125) by the mechanistic target of rapamycin (mTOR) downstream kinase P70S6K and that this phosphorylation increases its ability to degrade HIF1α. mTOR blockade in hypoxia by REDD1 restrains P70S6K and unleashes PP2A phosphatase activity. Through its regulatory subunit B55α, PP2A directly dephosphorylates PHD2 on S125, resulting in a further reduction of PHD2 activity that ultimately boosts HIF1α accumulation. These events promote autophagy-mediated cell survival in colorectal cancer (CRC) cells. B55α knockdown blocks neoplastic growth of CRC cells in vitro and in vivo in a PHD2-dependent manner. In patients, CRC tissue expresses higher levels of REDD1, B55α, and HIF1α but has lower phospho-S125 PHD2 compared with a healthy colon. Our data disclose a mechanism of PHD2 regulation that involves the mTOR and PP2A pathways and controls tumor growth., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Overcoming resistance to chemotherapy is a major challenge in colorectal cancer (CRC) treatment, especially since the underlying molecular mechanisms remain unclear. We show that silencing of the prolyl hydroxylase domain protein PHD1, but not PHD2 or PHD3, prevents p53 activation upon chemotherapy in different CRC cell lines, thereby inhibiting DNA repair and favoring cell death. Mechanistically, PHD1 activity reinforces p53 binding to p38α kinase in a hydroxylation-dependent manner. Following p53-p38α interaction and chemotherapeutic damage, p53 can be phosphorylated at serine 15 and thus activated. Active p53 allows nucleotide excision repair by interacting with the DNA helicase XPB, thereby protecting from chemotherapy-induced apoptosis. In accord with this observation, PHD1 knockdown greatly sensitizes CRC to 5-FU in mice. We propose that PHD1 is part of the resistance machinery in CRC, supporting rational drug design of PHD1-specific inhibitors and their use in combination with chemotherapy., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

Mutations or amplification of the MET proto-oncogene are involved in the pathogenesis of several tumours, which rely on the constitutive engagement of this pathway for their growth and survival. However, MET is expressed not only by cancer cells but also by tumour-associated stromal cells, although its precise role in this compartment is not well characterized. Here we show that MET is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor (HGF). Met deletion in mouse neutrophils enhances tumour growth and metastasis. This phenotype correlates with reduced neutrophil infiltration to both the primary tumour and metastatic sites. Similarly, Met is necessary for neutrophil transudation during colitis, skin rash or peritonitis. Mechanistically, Met is induced by tumour-derived tumour necrosis factor (TNF)-α or other inflammatory stimuli in both mouse and human neutrophils. This induction is instrumental for neutrophil transmigration across an activated endothelium and for inducible nitric oxide synthase production upon HGF stimulation. Consequently, HGF/MET-dependent nitric oxide release by neutrophils promotes cancer cell killing, which abates tumour growth and metastasis. After systemic administration of a MET kinase inhibitor, we prove that the therapeutic benefit of MET targeting in cancer cells is partly countered by the pro-tumoural effect arising from MET blockade in neutrophils. Our work identifies an unprecedented role of MET in neutrophils, suggests a potential 'Achilles' heel' of MET-targeted therapies in cancer, and supports the rationale for evaluating anti-MET drugs in certain inflammatory diseases.

Tumor-associated macrophages (TAM) are exposed to multiple microenvironmental cues in tumors, which collaborate to endow these cells with protumoral activities. Hypoxia, caused by an imbalance in oxygen supply and demand because of a poorly organized vasculature, is often a prominent feature in solid tumors. However, to what extent tumor hypoxia regulates the TAM phenotype in vivo is unknown. Here, we show that the myeloid infiltrate in mouse lung carcinoma tumors encompasses two morphologically distinct CD11b(hi)F4/80(hi)Ly6C(lo) TAM subsets, designated as MHC-II(lo) and MHC-II(hi) TAM, both of which were derived from tumor-infiltrating Ly6C(hi) monocytes. MHC-II(lo) TAM express higher levels of prototypical M2 markers and reside in more hypoxic regions. Consequently, MHC-II(lo) TAM contain higher mRNA levels for hypoxia-regulated genes than their MHC-II(hi) counterparts. To assess the in vivo role of hypoxia on these TAM features, cancer cells were inoculated in prolyl hydroxylase domain 2 (PHD2)-haplodeficient mice, resulting in better-oxygenated tumors. Interestingly, reduced tumor hypoxia did not alter the relative abundance of TAM subsets nor their M2 marker expression, but specifically lowered hypoxia-sensitive gene expression and angiogenic activity in the MHC-II(lo) TAM subset. The same observation in PHD2(+/+) → PHD2(+/-) bone marrow chimeras also suggests organization of a better-oxygenized microenvironment. Together, our results show that hypoxia is not a major driver of TAM subset differentiation, but rather specifically fine-tunes the phenotype of M2-like MHC-II(lo) TAM.

Ewing sarcoma is one of the most frequent bone cancers in adolescence. Although multidisciplinary therapy has improved the survival rate for localized tumors, a critical step is the development of new drugs to improve the long-term outcome of recurrent and metastatic disease and to reduce side effects of conventional therapy. Here, we show that the small molecule reactivation of p53 and induction of tumor cell apoptosis (RITA, NSC652287) is highly effective in reducing growth and tumorigenic potential of Ewing sarcoma cell lines. These effects occur both in the presence of wt-p53 as well as of mutant or truncated forms of p53, or in its absence, suggesting the presence of additional targets in this tumor histotype. Further experiments provided evidence that RITA modulates an important oncogenic mark of these cell lines, insulin-like growth factor receptor 1 (IGF-1R). Particularly, RITA causes downregulation of IGF-1R protein levels. MDM2 degradative activity is involved in this phenomenon. Indeed, inhibition of MDM2 function by genetic or pharmacologic approaches reduces RITA sensitivity of Ewing sarcoma cell lines. Overall, these data suggest that in the cell context of Ewing sarcoma, RITA may adopt additional mechanism of action besides targeting p53, expanding its field of application. Noteworthy, these results envisage the promising utilization of RITA or its derivative as a potential treatment for Ewing sarcomas., (©2012 AACR)