A phenotypic screening approach to target p60AmotL2-expressing invasive cancer cells.

BACKGROUND: Tumor cells have the ability to invade and form small clusters that protrude into adjacent tissues, a phenomenon that is frequently observed at the periphery of a tumor as it expands into healthy tissues. The presence of these clusters is linked to poor prognosis and has proven challenging to treat using conventional therapies. We previously reported that p60AmotL2 expression is localized to invasive colon and breast cancer cells. In vitro, p60AmotL2 promotes epithelial cell invasion by negatively impacting E-cadherin/AmotL2-related mechanotransduction. METHODS: Using epithelial cells transfected with inducible p60AmotL2, we employed a phenotypic drug screening approach to find compounds that specifically target invasive cells. The phenotypic screen was performed by treating cells for 72 h with a library of compounds with known antitumor activities in a dose-dependent manner. After assessing cell viability using CellTiter-Glo, drug sensitivity scores for each compound were calculated. Candidate hit compounds with a higher drug sensitivity score for p60AmotL2-expressing cells were then validated on lung and colon cell models, both in 2D and in 3D, and on colon cancer patient-derived organoids. Nascent RNA sequencing was performed after BET inhibition to analyse BET-dependent pathways in p60AmotL2-expressing cells. RESULTS: We identified 60 compounds that selectively targeted p60AmotL2-expressing cells. Intriguingly, these compounds were classified into two major categories: Epidermal Growth Factor Receptor (EGFR) inhibitors and Bromodomain and Extra-Terminal motif (BET) inhibitors. The latter consistently demonstrated antitumor activity in human cancer cell models, as well as in organoids derived from colon cancer patients. BET inhibition led to a shift towards the upregulation of pro-apoptotic pathways specifically in p60AmotL2-expressing cells. CONCLUSIONS: BET inhibitors specifically target p60AmotL2-expressing invasive cancer cells, likely by exploiting differences in chromatin accessibility, leading to cell death. Additionally, our findings support the use of this phenotypic strategy to discover novel compounds that can exploit vulnerabilities and specifically target invasive cancer cells.

Pyruvate dehydrogenase kinase regulates vascular inflammation in atherosclerosis and increases cardiovascular risk.

AIMS: Recent studies have revealed a close connection between cellular metabolism and the chronic inflammatory process of atherosclerosis. While the link between systemic metabolism and atherosclerosis is well established, the implications of altered metabolism in the artery wall are less understood. Pyruvate dehydrogenase kinase (PDK)-dependent inhibition of pyruvate dehydrogenase (PDH) has been identified as a major metabolic step regulating inflammation. Whether the PDK/PDH axis plays a role in vascular inflammation and atherosclerotic cardiovascular disease remains unclear. METHODS AND RESULTS: Gene profiling of human atherosclerotic plaques revealed a strong correlation between PDK1 and PDK4 transcript levels and the expression of pro-inflammatory and destabilizing genes. Remarkably, the PDK1 and PDK4 expression correlated with a more vulnerable plaque phenotype, and PDK1 expression was found to predict future major adverse cardiovascular events. Using the small-molecule PDK inhibitor dichloroacetate (DCA) that restores arterial PDH activity, we demonstrated that the PDK/PDH axis is a major immunometabolic pathway, regulating immune cell polarization, plaque development, and fibrous cap formation in Apoe-/- mice. Surprisingly, we discovered that DCA regulates succinate release and mitigates its GPR91-dependent signals promoting NLRP3 inflammasome activation and IL-1ß secretion by macrophages in the plaque. CONCLUSIONS: We have demonstrated for the first time that the PDK/PDH axis is associated with vascular inflammation in humans and particularly that the PDK1 isozyme is associated with more severe disease and could predict secondary cardiovascular events. Moreover, we demonstrate that targeting the PDK/PDH axis with DCA skews the immune system, inhibits vascular inflammation and atherogenesis, and promotes plaque stability features in Apoe-/- mice. These results point toward a promising treatment to combat atherosclerosis.

Genetic Deficiency of Indoleamine 2,3-dioxygenase Aggravates Vascular but Not Liver Disease in a Nonalcoholic Steatohepatitis and Atherosclerosis Comorbidity Model.

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that increases cardiovascular disease risk. Indoleamine 2,3-dioxygenase-1 (IDO1)-mediated tryptophan (Trp) metabolism has been proposed to play an immunomodulatory role in several diseases. The potential of IDO1 to be a link between NASH and cardiovascular disease has never been investigated. Using Apoe-/-and Apoe-/-Ido1-/- mice that were fed a high-fat, high-cholesterol diet (HFCD) to simultaneously induce NASH and atherosclerosis, we found that Ido1 deficiency significantly accelerated atherosclerosis after 7 weeks. Surprisingly, Apoe-/-Ido1-/- mice did not present a more aggressive NASH phenotype, including hepatic lipid deposition, release of liver enzymes, and histopathological parameters. As expected, a lower L-kynurenine/Trp (Kyn/Trp) ratio was found in the plasma and arteries of Apoe-/-Ido1-/- mice compared to controls. However, no difference in the hepatic Kyn/Trp ratio was found between the groups. Hepatic transcript analyses revealed that HFCD induced a temporal increase in tryptophan 2,3-dioxygenase (Tdo2) mRNA, indicating an alternative manner to maintain Trp degradation during NASH development in both Apoe-/- and Apoe-/-Ido1-/mice-. Using HepG2 hepatoma cell and THP1 macrophage cultures, we found that iron, TDO2, and Trp degradation may act as important mediators of cross-communication between hepatocytes and macrophages regulating liver inflammation. In conclusion, we show that Ido1 deficiency aggravates atherosclerosis, but not liver disease, in a newly established NASH and atherosclerosis comorbidity model. Our data indicate that the overexpression of TDO2 is an important mechanism that helps in balancing the kynurenine pathway and inflammation in the liver, but not in the artery wall, which likely determined disease outcome in these two target tissues.

Canonical WNT signaling-dependent gating of MYC requires a noncanonical CTCF function at a distal binding site.

Abnormal WNT signaling increases MYC expression in colon cancer cells in part via oncogenic super-enhancer-(OSE)-mediated gating of the active MYC to the nuclear pore in a poorly understood process. We show here that the principal tenet of the WNT-regulated MYC gating, facilitating nuclear export of the MYC mRNA, is regulated by a CTCF binding site (CTCFBS) within the OSE to confer growth advantage in HCT-116 cells. To achieve this, the CTCFBS directs the WNT-dependent trafficking of the OSE to the nuclear pore from intra-nucleoplasmic positions in a stepwise manner. Once the OSE reaches a peripheral position, which is triggered by a CTCFBS-mediated CCAT1 eRNA activation, its final stretch (≤0.7 µm) to the nuclear pore requires the recruitment of AHCTF1, a key nucleoporin, to the CTCFBS. Thus, a WNT/ß-catenin-AHCTF1-CTCF-eRNA circuit enables the OSE to promote pathological cell growth by coordinating the trafficking of the active MYC gene within the 3D nuclear architecture.

Disruption of GPR35 Signaling in Bone Marrow-Derived Cells Does Not Influence Vascular Inflammation and Atherosclerosis in Hyperlipidemic Mice.

G-protein-coupled receptor-35 (GPR35) has been identified as a receptor for the tryptophan metabolite kynurenic acid (KynA) and suggested to modulate macrophage polarization in metabolic tissues. Whether GPR35 can influence vascular inflammation and atherosclerosis has however never been tested. Lethally irradiated LdlrKO mice were randomized to receive GPR35KO or wild type (WT) bone marrow transplants and fed a high cholesterol diet for eight weeks to develop atherosclerosis. GPR35KO and WT chimeric mice presented no difference in the size of atherosclerotic lesions in the aortic arch (2.37 ± 0.58% vs. 1.95 ± 0.46%, respectively) or in the aortic roots (14.77 ± 3.33% vs. 11.57 ± 2.49%, respectively). In line with these data, no changes in the percentage of VCAM-1+, IAb + cells, and CD3+ T cells, as well as alpha smooth muscle cell actin expression, was observed between groups. Interestingly, the GPR35KO group presented a small but significant increase in CD68+ macrophage infiltration in the plaque. However, in vitro culture experiments using bone marrow-derived macrophages from both groups indicated that GPR35 plays no role in modulating the secretion of major inflammatory cytokines. Our study indicates that GPR35 expression does not play a direct role in macrophage activation, vascular inflammation, and the development of atherosclerosis.

Insulina modula migração celular e a secreção de citocinas na vigência da infecção pulmonar por Paracoccidioides brasiliensis em camundongos diabéticos e não-diabéticos / Insulin modulates cell migration and cytokines secretion during pulmonary infection caused by Paracoccidioides brasiliensis in diabetic and non-diabetic mice

Diabetes mellitus (DM) compreende um conjunto de doenças metabólicas de grande importância e incidência mundial. Nele, o DM do tipo 1 é caracterizado pela destruição de células pancreáticas produtoras de insulina, e dentre seus sintomas, a disfunção imunológica relacionada à falta de insulina foi observada por diversos estudos, descrevendo pacientes diabéticos como mais susceptíveis a infecções e complicações decorrentes destas. Paracoccidioidomicose (PCM) é uma enfermidade sistêmica causada por fungos da espécie Paracoccidioides sp., bastante importante no Brasil e endêmica em toda a América Latina. Este trabalho utiliza um modelo de carência relativa de insulina (DM experimental) para estudar a intervenção da insulina em um modelo de micose pulmonar causada por P. brasiliensis, analisando o processo de migração celular (expressão de moléculas de adesão por imunohistoquímica e fenótipo dos leucócitos do pulmão por citometria de fluxo), os mecanismos moleculares (produção/liberação de citocinas por cytometric bead array), intracelulares (vias de sinalização por Western blot), e a atividade fagocítica e microbicida dos macrófagos alveolares. Em resultados observamos que, comparados aos não-diabéticos, camundongos tornados diabéticos apresentam maior susceptibilidade evidenciada por menor atividade fagocítica e reduzidas secreções de interferon-γ e de interleucina-12 na fase inicial da inflamação, que leva a uma resposta menos efetiva com menor expressão de molécula de adesão de células vasculares, reduzidas populações de linfócitos TCD4+, TCD8+, células natural killer, culminando em inflamação crônica resultante da proliferação aumentada do fungo nos pulmões (aumento de interferon-γ e fator necrótico tumoral-ß). Vemos ainda que o tratamento de insulina em animais diabéticos restaurou as secreções de citocinas pró-inflamatórias e a atividade fagocítica de macrófagos em 24 horas de infecção, e aumentou a celularidade, a expressão de moléculas de adesão de células vasculares-1 e restaurou as populações de linfócitos B, de células natural killer e de células coestimuladas por CD80, além de reduzir a inflamação crônica no pulmão. Estes dados em conjunto nos permitem inferir que a insulina modulou o ambiente inflamatório de animais tornados diabéticos de formas diferentes em estágios iniciais e tardios da infecção pelo isolado Pb18 do Paracoccidioides brasiliensis

Diabetes mellitus comprehends a group of metabolic diseases of great importance and incidence worldwide. Type 1 diabetes mellitus is characterized by destruction of insulin producing-pancreatic cells and, among its symptoms, an impaired immunological function has been observed in many studies having diabetic patients described as more susceptible to infections and complications resulted of them. Paracoccidioidomycosis is a systemic disease caused by fungi of Paracoccidioides spp. , also of great importance in Brazil and endemic in the whole Latin America. This work uses a model of experimental T1DM to investigate the intervention of insulin in a model of murine PCM induced by Paracoccidioides brasiliensis, analyzing the process of cell migration (adhesion molecules expression, leukocyte phenotyping), molecular mechanisms (production and secretion of cytokines), intracellular mechanisms (signaling pathways) and phagocytic and microbicidal activities in alveolar macrophages. In results, compared to controls, we observed higher susceptibility in diabetic mice to PCM, evidenced by reduced phagocytic activity and reduced levels of interferon-γ and interleukin-12 on initial stages of infection, and a less effective inflammation with lesser expression of adhesion molecules, reduced migration of TCD4+, TCD8+, NK cells and B lymphocytes, resulting in chronic inflammation caused by higher fungal proliferation in lungs (higher interferon-γ and tumours necrosis factor-α levels). In addition, we saw treatment with insulin in diabetic animals restored secretion of pro-inflammatory cytokines and phagocytic activity on early stages and allowed higher cellularity, higher expression of vascular cells adhesion molecule-1 and restored populations of B lymphocytes, NK cells and the expression of costimularoty molecule CD80, also reducing the chronic inflammation in lungs. Taken together, these data lead us to suggest insulin modulated the inflammatory microenvironment in lungs of mice rendered diabetic, in different forms on earlier and later stages of an infection by Pb18 isolate

Insulin Modulates Inflammatory Cytokine Release in Acute Stages and Augments Expression of Adhesion Molecules and Leukocytes in Lungs on Chronic Stages of Paracoccidioidomycosis.

Type 1 diabetesmellitus (T1D) is caused by partial destruction of the insulin-producing beta cells in the pancreas and is a major issue for public health care worldwide. Reduced or impaired immunological responses, which render patients more susceptible to infections, have been observed in T1D, and this dysfunction is often related to a lack of insulin in the blood. Paracoccidioidomycosis is an important systemic mycosis endemic in Latin America. To evaluate the effects of T1D on this fungal infection and the modulatory effects of insulin, we induced diabetes in C57Bl/6 male mice (alloxan, 60 mg/kg), infected the mice (Pb18, 1 x 106 cells), and treated the mice with neutral protamine Hagedorn (NPH) insulin (2 IU/600 mg/dL blood glucose). Twenty-four hours after infection, infected diabetic mice showed reduced secretion of interferon (IFN)-γ and interleukine (IL)-12 p70 compared to infected nondiabetic controls. On the 45th day of infection, infected diabetic mice presented higher IFN-γ levels, a higher tumor necrosis factor (TNF)-α:IL-10 ratio, and lower adhesion molecule expression levels than nondiabetic mice. In the in vitro experiments, alveolar macrophages from diabetic animals showed reduced phagocytic activity compared to those from control animals at 4, 12, and 24 h. In infected diabetic mice, treatment with insulin restored IL-12 p70 levels at 24 h of infection, reduced IFN-γ levels and the TNF-α:IL-10 ratio at 45 days, and restored vascular cell adhesion molecule (VCAM)-1 expression in pulmonary blood vessels, and this treatment reduced the diminished phosphorylation of extracellular signal-regulated kinases (ERK) and increased nuclear factor-kappa-B(iκb)-α and jun amino-terminal kinases (JNK) p46 levels in infected nondiabetic mice. In addition, insulin promoted increased phagocytic activity in the alveolar macrophages of diabetic mice. These data suggest that T1D mice are more susceptible to Pb18 infection and that insulin modulates this inflammation in diabetic mice by augmenting the expression of adhesion molecules and leukocytes in the lungs and by reducing chronic inflammation.

Insulin Modulates the Immune Cell Phenotype in Pulmonary Allergic Inflammation and Increases Pulmonary Resistance in Diabetic Mice.

Introduction: Reports have shown that the onset of diabetes mellitus (DM) in patients previously diagnosed with asthma decreases asthmatic symptoms, whereas insulin aggravates asthma. The present study evaluated the modulatory effect of insulin on the development of allergic airway inflammation in diabetic mice. Materials and Methods: To evaluate the effects of relative insulin deficiency, an experimental model of diabetes was induced by a single dose of alloxan (50 mg/kg, i.v.). After 10 days, the mice were sensitized with ovalbumin [OVA, 20 µg and 2 mg of Al(OH)3, i.p.]. A booster immunization was performed 6 days after the first sensitization [20 µg of OVA and 2 mg of Al(OH)3, i.p.]. The OVA challenge (1 mg/mL) was performed by daily nebulization for 7 days. Diabetic animals were treated with multiple doses of neutral protamine Hagedorn (NPH) before each challenge with OVA. The following parameters were measured 24 h after the last challenge: (a) the levels of p38 MAP kinase, ERK 1/2 MAP kinases, JNK, STAT 3, and STAT 6 in lung homogenates; (b) the serum profiles of immunoglobulins IgE and IgG1; (c) the concentrations of cytokines (IL-4, IL-5, IL-10, IL-13, TNF-α, VEGF, TGF-ß, and IFN-γ) in lung homogenates; (d) cells recovered from the bronchoalveolar lavage fluid (BALF); (e) the profiles of immune cells in the bone marrow, lung, thymus, and spleen; and (f) pulmonary mechanics using invasive (FlexiVent) and non-invasive (BUXCO) methods. Results: Compared to non-diabetic OVA-challenged mice, OVA-challenged diabetic animals showed decreases in ERK 1 (2-fold), ERK 2 (7-fold), JNK (phosphor-54) (3-fold), JNK/SAPK (9-fold), STAT3 (4-fold), the levels of immunoglobulins, including IgE (1-fold) and IgG1 (3-fold), cytokines, including Th2 profile cytokines such as IL-4 (2-fold), IL-5 (2-fold), IL-13 (4-fold), TNF-α (2-fold), VEGF (2-fold), and TGF-ß (2-fold), inflammatory infiltrates (14-fold), T cells, NK cells, B cells and eosinophils in the bone marrow, lung, thymus and spleen, and airway hyperreactivity. STAT6 was absent, and no eosinophilia was observed in BALF. Insulin treatment restored all parameters. Conclusion: The data suggested that insulin modulates immune cell phenotypes and bronchial hyperresponsiveness in the development of allergic airway inflammation in diabetic mice.

Insulin Modulates Paracoccidioides brasiliensis-Induced Inflammation by Restoring the Populations of NK Cells, Dendritic Cells, and B Lymphocytes in Lungs.

Paracoccidioidomycosis, a key issue for Brazilian health service, can be aggravated in patients with impaired immunological responses, such as diabetic patients. We evaluated the role of insulin in inflammatory parameters in diabetic and nondiabetic mice using a systemic mycosis Paracoccidioides brasiliensis (Pb) model. Diabetic C57BL-6 mice and controls were infected with Pb18 and treated with insulin for 12 days prior to experiments. After 55 days, infected diabetic mice exhibited fewer leukocytes in both peritoneal lavage fluid (PeLF) and bronchoalveolar lavage fluid and reduced secretion of interleukin- (IL-) 6 in lungs. In addition, diabetic mice presented a reduced influx of TCD4+ cells, TCD8+ cells, B lymphocytes, NK cells, and dendritic cells compared to control infected groups. Insulin treatment restored the leukocyte number in PeLF and restored the presence of B lymphocytes, dendritic cells, and NK cells in lungs of diabetic animals. The data suggest that diabetic mice present impaired immunological response to Pb18 infection and insulin modulates inflammation by reducing IL-6 levels in lung and CINC-1 levels in spleen and liver homogenates, restoring leukocyte concentrations in PeLF and also restoring populations of dendritic cells and B lymphocytes in lungs of diabetic mice, permitting the host to better control the infection.

Cytokine and Adhesion Molecule Expression Induced by Different Strains of Staphylococcus aureus in Type 1 Diabetic Rats: Role of Insulin.

Introduction:Staphylococcus aureus may provoke peritonitis and death, especially in immunocompromized individuals such as diabetic patients. We evaluated the role of insulin in S. aureus-induced peritoneal infection in diabetic and non-diabetic rats. Materials/Methods: Alloxan-diabetic male Wistar rats and their respective controls received intraperitoneal injections of different strains of S. aureus or sterile phosphate-buffered saline. After 3 days of infection, the first set of diabetic and non-diabetic rats received 4 and 1 IU, respectively, of neutral protamine Hagedorn insulin and were analyzed 8 h later. The second set of diabetic and non-diabetic rats received 4 and 1 IU, respectively, of insulin 2 h before intraperitoneal infection and a half dose of insulin at 5 p.m. for the next 2 days and were analyzed 16 h later. The following measurements were performed: (a) number of cells in the peritoneal lavage fluid (PeLF), white blood cell count, and blood glucose; (b) serum insulin and corticosterone; (c) cytokine levels in the PeLF; (d) expression of adhesion molecules in the vascular endothelium; and (e) microbicidal activity. Results: Diabetic rats showed an increased number of polymorphonuclear leukocytes (PMNs) and increased concentrations of CINC-1, IL-4, and IFN-γ in the PeLF after infection with the ATCC 25923 or N315 αHL+ strain. The mesenteric expression of PECAM-1 was increased after infection with the N315 HLA+ strain. ICAM-1 expression was increased with ATCC infection. Treatment of diabetic rats with a single dose of insulin restored CINC-1 levels in the PeLF for both strains; however, PMN migration, IL-4, and IFN-γ were restored in rats infected with the ATCC strain, whereas the PeLF concentrations of CINC-2, IL-1ß, and IL-4 were increased in N315-infected animals. Insulin restored PMN migration and CINC-2 levels in the PeLF in ATCC-infected rats. After multiple treatments with insulin, the levels of IL-1ß, IL-6, and IFN-γ were increased in the PeLF of diabetic rats after infection with either strain, and CINC-2 levels were restored in N315-infected animals. Conclusion: These results suggest that insulin distinctively modulates cytokine production or release, PMN leukocyte migration, and adhesion molecule expression during the course of peritonitis induced by different strains of S. aureus.

Insulin Modulates Cytokine Release, Collagen and Mucus Secretion in Lung Remodeling of Allergic Diabetic Mice.

INTRODUCTION: The role of insulin in lung remodeling in a model of asthma in healthy and diabetic mice was evaluated. MATERIAL AND METHODS: Diabetic male BALB/c mice (alloxan, 50 mg/kg, intravenous) and controls were sensitized by subcutaneous (s.c.) injection of ovalbumin (OA, 20 µg) in aluminum hydroxide (Al(OH)3, 2 mg) 10 days after the alloxan injection and received the same dose 12 days later. Six days after the last sensitization, animals were nebulized with OA solution for 7 days. The first set of diabetic and control mice received 2 and 1 IU, respectively, of s.c. neutral protamine Hagedorn (NPH) insulin and were analyzed 8 h later. The second set of diabetic and control mice received 2 and 1 IU, respectively, of insulin 12 h before the OA challenge and half doses of insulin 2 h before each the seven OA challenges. Twenty-four hours after the last challenge, the following analyses were performed: (a) quantification of the cells in the bronchoalveolar lavage fluid (BALF), the white cell count, and blood glucose; (b) morphological analysis of lung tissues by hematoxylin and eosin staining; (c) quantification of collagen deposition in lung tissues and mucus by morphometric analysis of histological sections stained with Masson's trichrome and periodic acid-Schiff (PAS), respectively; and (d) quantification of the cytokine concentrations (IL-4, IL-5, and IL-13) in the BALF supernatant. RESULTS: Compared to controls, diabetic mice had significantly reduced inflammatory cells (81%) in the BALF, no eosinophils in the BALF and peripheral blood and reduced collagen deposition and mucus in the lungs. BALF concentrations of IL-4 (48%) and IL-5 (31%) decreased and IL-13 was absent. A single dose of insulin restored peripheral blood eosinophils and BALF mononuclear cells but not BALF eosinophils, collagen deposition, and mucus levels. However, multiple doses of insulin restored both total cells and eosinophils in the BALF and peripheral blood, BALF cytokines, and collagen deposition and mucus secretion into the lungs. CONCLUSION: The results suggest that insulin modulates the production/release of cytokines, cell migration, deposition of collagen, and mucus secretion in lung remodeling of a mouse model of asthma.