A microbiota-modulated checkpoint directs immunosuppressive intestinal T cells into cancers.

Antibiotics (ABX) compromise the efficacy of programmed cell death protein 1 (PD-1) blockade in cancer patients, but the mechanisms underlying their immunosuppressive effects remain unknown. By inducing the down-regulation of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the ileum, post-ABX gut recolonization by Enterocloster species drove the emigration of enterotropic α4ß7+CD4+ regulatory T 17 cells into the tumor. These deleterious ABX effects were mimicked by oral gavage of Enterocloster species, by genetic deficiency, or by antibody-mediated neutralization of MAdCAM-1 and its receptor, α4ß7 integrin. By contrast, fecal microbiota transplantation or interleukin-17A neutralization prevented ABX-induced immunosuppression. In independent lung, kidney, and bladder cancer patient cohorts, low serum levels of soluble MAdCAM-1 had a negative prognostic impact. Thus, the MAdCAM-1-α4ß7 axis constitutes an actionable gut immune checkpoint in cancer immunosurveillance.

CDK4/6 Inhibition Enhances the Efficacy of Standard Chemotherapy Treatment in Malignant Pleural Mesothelioma Cells.

BACKGROUND: The loss of the CDKN2A/ARF (cyclin-dependent kinase inhibitor 2A/alternative reading frame) gene is the most common alteration in malignant pleural mesothelioma (MPM), with an incidence of about 70%, thus representing a novel target for mesothelioma treatment. In the present study, we evaluated the antitumor potential of combining the standard chemotherapy regimen used for unresectable MPM with the CDK4/6 (cyclin-dependent kinase 4 or 6) inhibitor abemaciclib. METHODS: Cell viability, cell death, senescence, and autophagy induction were evaluated in two MPM cell lines and in a primary MPM cell culture. RESULTS: The simultaneous treatment of abemaciclib with cisplatin and pemetrexed showed a greater antiproliferative effect than chemotherapy alone, both in MPM cell lines and in primary cells. This combined treatment induced cellular senescence or autophagic cell death, depending on the cell type. More in detail, the induction of cellular senescence was related to the increased expression of p21, whereas autophagy induction was due to the impairment of the AKT/mTOR signaling. Notably, the effect of the combination was irreversible and no resumption in tumor cell proliferation was observed after drug withdrawal. CONCLUSION: Our results demonstrated the therapeutic potential of CDK4/6 inhibitors in combination with chemotherapy for the treatment of MPM and are consistent with the recent positive results in the MiST2 arm in abemaciclib-treated patients.

CDK4/6 inhibitors improve the anti-tumor efficacy of lenvatinib in hepatocarcinoma cells.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer with a poor prognosis and limited treatment options. Considering that alterations of the CDK4/6-cyclin D-Rb pathway occur frequently in HCC, we tested the efficacy of two CDK4/6 inhibitors, abemaciclib and ribociclib, in combination with lenvatinib, a multi-kinase inhibitor approved as first-line therapy for advanced HCC, in a panel of HCC Rb-expressing cell lines. The simultaneous drug combinations showed a superior anti-proliferative activity as compared with single agents or sequential schedules of treatment, either in short or in long-term experiments. In addition, the simultaneous combination of abemaciclib with lenvatinib reduced 3D cell growth, and impaired colony formation and cell migration. Mechanistically, these growth-inhibitory effects were associated with a stronger down-regulation of c-myc protein expression. Depending on the HCC cell model, reduced activation of MAPK, mTORC1/p70S6K or src/FAK signaling was also observed. Abemaciclib combined with lenvatinib arrested the cells in the G1 cell cycle phase, induced p21 accumulation, and promoted a stronger increase of cellular senescence, associated with elevation of ß-galactosidase activity and accumulation of ROS, as compared with single treatments. After drug withdrawal, the capacity of forming colonies was significantly impaired, suggesting that the anti-tumor efficacy of abemaciclib and lenvatinib combination was persistent. Our pre-clinical results demonstrate the effectiveness of the simultaneous combination of CDK4/6 inhibitors with lenvatinib in HCC cell models, suggesting that this combination may be worthy of further investigation as a therapeutic approach for the treatment of advanced HCC.

Immunotherapeutic Approaches in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare and aggressive malignant disease affecting the mesothelium, commonly associated to asbestos exposure. The current therapeutic actions, based on cisplatin/pemetrexed treatment, are limited due to the late stage at which most patients are diagnosed and to the intrinsic chemo-resistance of the tumor. Another relevant point is the absence of approved therapies in the second line setting following progression of MPM after chemotherapy. Considering the poor prognosis of the disease and the fact that the incidence of this tumor is expected to increase in the next decade, novel therapeutic approaches are urgently needed. In the last few years, several studies have investigated the efficacy and safety of immune-checkpoint inhibitors (ICIs) in the treatment of unresectable advanced MPM, and a number of trials with immunotherapeutic agents are ongoing in both first line and second line settings. In this review, we describe the most promising emerging immunotherapy treatments for MPM (ICIs, engineered T cells to express chimeric antigen receptors (CARs), dendritic cells (DCs) vaccines), focusing on the biological and immunological features of this tumor as well as on the issues surrounding clinical trial design.

New Imidazo[2,1-b][1,3,4]Thiadiazole Derivatives Inhibit FAK Phosphorylation and Potentiate the Antiproliferative Effects of Gemcitabine Through Modulation of the Human Equilibrative Nucleoside Transporter-1 in Peritoneal Mesothelioma.

BACKGROUND/AIM: A new class of imidazo[2,1-b][1,3,4]thiadiazole compounds have recently been evaluated as inhibitors of phosphorylation of focal adhesion kinase (FAK) in pancreatic cancer. FAK is overexpressed in mesothelioma and has recently emerged as an interesting target for the treatment of this disease. MATERIALS AND METHODS: Ten imidazo[2,1-b][1,3,4]thiadiazole compounds characterized by indole bicycle and a thiophene ring, were evaluated for their cytotoxic activity in two primary cell cultures of peritoneal mesothelioma, MesoII and STO cells. RESULTS: Compounds 1a and 1b showed promising antitumor activity with IC50 values in the range of 0.59 to 2.81 µM in both cell lines growing as monolayers or as spheroids. Their antiproliferative and antimigratory activity was associated with inhibition of phospho-FAK, as detected by a specific ELISA assay in STO cells. Interestingly, these compounds potentiated the antiproliferative activity of gemcitabine, and these results might be explained by the increase in the mRNA expression of the key gemcitabine transporter human equilibrative nucleoside transporter-1 (hENT-1). CONCLUSION: These promising results support further studies on new imidazo[2,1-b][1,3,4]thiadiazole compounds as well as on the role of both FAK and hENT-1 modulation in order to develop new drug combinations for peritoneal mesothelioma.

Dual Inhibition of CDK4/6 and PI3K/AKT/mTOR Signaling Impairs Energy Metabolism in MPM Cancer Cells.

Background: Malignant pleural mesothelioma (MPM) is an aggressive malignancy associated to asbestos exposure. One of the most frequent genetic alteration in MPM patients is CDKN2A/ARF loss, leading to aberrant activation of the Rb pathway. In MPM cells, we previously demonstrated the therapeutic efficacy of targeting this signaling with the CDK4/6 inhibitor palbociclib in combination with PI3K/mTOR inhibitors. Here, we investigated whether such combination may have an impact on cell energy metabolism. Methods: The study was performed in MPM cells of different histotypes; metabolic analyses were conducted by measuring GLUT-1 expression and glucose uptake/consumption, and by SeaHorse technologies. Results: MPM cell models differed for their ability to adapt to metabolic stress conditions, such as glucose starvation and hypoxia. Independently of these differences, combined treatments with palbociclib and PI3K/mTOR inhibitors inhibited cell proliferation more efficaciously than single agents. The drugs alone reduced glucose uptake/consumption as well as glycolysis, and their combination further enhanced these effects under both normoxic and hypoxic conditions. Moreover, the drug combinations significantly impaired mitochondrial respiration as compared with individual treatments. These metabolic effects were mediated by the concomitant inhibition of Rb/E2F/c-myc and PI3K/AKT/mTOR signaling. Conclusions: Dual blockade of glycolysis and respiration contributes to the anti-tumor efficacy of palbociclib-PI3K/mTOR inhibitors combination.

Tonsillectomy efficacy in children with PFAPA syndrome is comparable to the standard medical treatment: a long-term observational study.

OBJECTIVES: Tonsillectomy has recently been suggested as an effective treatment for PFAPA syndrome but little is known about its long-term efficacy. We compared the clinical features and the long-term outcome of a large cohort of patients with PFAPA syndrome treated with tonsillectomy or with standard medical treatment. METHODS: We conducted a retrospective study on patients with PFAPA syndrome followed at a tertiary care centre from January 1993 to August 2010. Clinical characteristics and laboratory parameters were evaluated at onset and during the follow-up. Disease outcomes of patients who underwent tonsillectomy and of those treated with medical therapy (NSAIDs, prednisone) were compared. Clinical remission on medication (CRM) was considered the persistence of fever attacks which were well controlled by medical therapy, clinical remission (CR) was defined as the absence of fever attacks, without any treatment, for more than 12 months. RESULTS: 275 patients with PFAPA syndrome, 59.6% males, aged 27.9 months at onset and followed for mean 54.5 months, entered the study. CR was reported in 59.6% of the patients and was significantly less frequent in those with positive family history for PFAPA (46.4% vs. 66.1%, p=0.003). 27/41 patients (65.9%), responded to tonsillectomy and this result was comparable with that observed in those treated with medical therapy (59.1%, p=0.51). Disease duration, age at remission or presence of associated symptoms were not significantly different in both groups. No predictors of tonsillectomy failure were found. CONCLUSIONS: In a large cohort of patients with PFAPA syndrome, tonsillectomy efficacy was comparable to the standard medical treatment.

Endogenous cannabinoid system regulates intestinal barrier function in vivo through cannabinoid type 1 receptor activation.

The deleterious effects of stress on the gastrointestinal tract seem to be mainly mediated by the induction of intestinal barrier dysfunction and subsequent subtle mucosal inflammation. Cannabinoid 1 receptor (CB1R) is expressed in the mammalian gut under physiological circumstances. The aim of this investigation is to study the possible role of CB1R in the maintenance of mucosal homeostasis after stress exposure. CB1R knockout mice (CB1R(-/-)) and their wild-type (WT) counterparts were exposed to immobilization and acoustic (IA) stress for 2 h per day during 4 consecutive days. Colonic protein expression of the inducible forms of the nitric oxide synthase and cyclooxygenase (NOS2 and COX2), IgA production, permeability to (51)Cr-EDTA, and bacterial translocation to mesenteric lymph nodes were evaluated. Stress exposure induced greater expression of proinflammatory enzymes NOS2 and COX2 in colonic mucosa of CB1R(-/-) mice when compared with WT animals. These changes were related with a greater degree of colonic barrier dysfunction in CB1R(-/-) animals determined by 1) a significantly lower IgA secretion, 2) higher paracellular permeability to (51)Cr-EDTA, and 3) higher bacterial translocation, both under basal conditions and after IA stress exposure. Pharmacological antagonism with rimonabant reproduced stress-induced increase of proinflammatory enzymes in the colon described in CB1R(-/-) mice. In conclusion, CB1R exerts a protective role in the colon in vivo through the regulation of intestinal secretion of IgA and paracellular permeability. Pharmacological modulation of cannabinoid system within the gastrointestinal tract might be therapeutically useful in conditions on which intestinal inflammation and barrier dysfunction takes place after exposure to stress.

Chronic blockade of cannabinoid CB2 receptors induces anxiolytic-like actions associated with alterations in GABA(A) receptors.

BACKGROUND AND PURPOSE: The aim of this study was to explore the effects of CB(2) receptor agonist and antagonist in the regulation of anxiety-like behaviours. EXPERIMENTAL APPROACHES: Effects of acute and chronic treatment with the CB(2) receptor agonist JWH133 and CB(2) receptor antagonist AM630 were evaluated in the light-dark box (LDB) and elevated plus maze (EPM) tests in Swiss ICR mice. CB(2) receptor, GABA(A) α(2) and GABA(A) γ(2) gene and protein expression in the cortex and amygdala of mice chronically treated with JWH133 or AM630 were examined by RT-PCR and Western blot. Effects of chronic AM630 treatment were evaluated in spontaneously anxious DBA/2 mice in LDB. KEY RESULTS: Acute JWH133 treatment failed to produce any effect. Acute AM630 treatment increased anxiety and was blocked by pre-treatment with JWH133. Chronic JWH133 treatment increased anxiety-like behaviour whereas chronic AM630 treatment was anxiolytic in LDB and EPM tests. Chronic AM630 treatment increased gene and reduced protein expression of CB(2) receptors, GABA(A) α(2) and GABA(A) γ(2) in cortex and amygdala. Chronic JWH133 treatment resulted in opposite gene and protein alterations. In addition, chronic AM630 administration decreased the anxiety of DBA/2 mice in the LDB test. CONCLUSIONS AND IMPLICATIONS: The opposing behavioural and molecular changes observed after chronic treatment with AM630 or JWH133 support the key role of CB(2) receptors in the regulation of anxiety. Indeed, the efficacy of AM630 in reducing the anxiety of the spontaneously anxious DBA/2 strain of mice strengthens the potential of the CB(2) receptor as a new target in the treatment of anxiety-related disorders.

Regulatory role of cannabinoid receptor 1 in stress-induced excitotoxicity and neuroinflammation.

Exposure to stress elicits excitoxicity and neuroinflammation in the brain, contributing to cell death and damage in stress-related neurological and neuropsychiatric diseases. The endocannabinoid system is present in stress-responsive neural circuits and has been proposed as an endogenous neuroprotective system activated in some neuropathological scenarios to restore homeostasis. To elucidate the possible regulatory role of cannabinoid receptor 1 (CB1) in stress-induced excitotoxicity and neuroinflammation, both genetic and pharmacological approaches were used alternatively: (1) wild-type (WT) and CB1 knockout mice (CB1-KO) were exposed to immobilization/acoustic stress (2 h/day for 4 days) and (2) to specifically activate CB1, the selective CB1 agonist Arachidonyl-2'-chloroethylamide (ACEA) (2.5 mg/kg) was intraperitoneally administered daily to some groups of animals. Stress exposure increased CB1 mRNA and protein expression in the prefrontal cortex of WT mice in a mechanism related to N-methyl-D-aspartate glutamate receptor activation. Daily ACEA pretreatment prevented stress-induced: (1) upregulation of CB1 mRNA and protein, (2) decrease in glutamate uptake and glutamate astroglial transporter excitatory amino acid transporter 2 expression, (3) increase in consecutive proinflammatory molecules, such as cytokines (tumor necrosis factor-α and MCP-1), nuclear factor kappa B, and enzymatic sources, such as inducible nitric oxide synthase (NOS-2) and cyclooxygenase-2 (COX-2), (4) increase in lipid peroxidation; although having no effect on plasma corticosterone. Interestingly, a possible related mechanism could be the positive ACEA modulation of the antiinflammatory pathway deoxyprostaglandin/peroxisome proliferator-activated receptor γ (15d-PGJ(2)/PPARγ). Conversely, KO animal experiments indicated that a lack of CB1 produces hypothalamic/pituitary/adrenal (HPA) axis dysregulation and exacerbates stress-induced excitotoxic/neuroinflammatory responses. These multifaceted neuroprotective effects suggest that CB1 activation could be a new therapeutic strategy against neurological/neuropsychiatric pathologies with HPA axis dysregulation and an excitotoxic/neuroinflammatory component in their pathophysiology.

Corticosterone basal levels and vulnerability to LPS-induced neuroinflammation in the rat brain.

To assess whether the individual differences on the brain response to lipopolysaccharide (LPS) are correlated with the individual differences in the hypothalamus-pituitary-adrenal axis basal activity, adult male outbred rats were injected i.p. with 1 mg/kg LPS and evaluated after 4 h. Basal (1 week before LPS) and post-LPS plasma corticosterone (CC) were measured (mean basal: 225+/-22 ng/mL at 15:00 h). Group H was assigned to animals with 33% higher levels of CC (>234 ng/mL) and group L to animals with 33% lower levels of CC (<167 ng/mL). The H group showed an 8.8 times less relative increase of CC after LPS than the L group as well as a reduced glucocorticoid receptor upregulation after LPS. In addition, H individuals present higher plasma levels of TNF-alpha and IL-1beta after LPS. Interestingly, these animals are more vulnerable to the accumulation of oxidative/nitrosative mediators in the brain (NF-kappaB, NOS-2 and COX-2). Concomitantly, H animals are less protected against LPS-induced neuroinflammation, since anti-inflammatory mediators, lipocalin-prostaglandinD2 synthase and peroxisome proliferator-activated gamma, are downregulated after LPS. These data demonstrate that CC plasma basal levels might be a relevant parameter for predicting the individual response to LPS.