Activation Of Intracellular Signaling Research Articles

Humanin activates integrin αV–TGFβ axis and leads to glioblastoma progression

The role of mitochondria peptides in the spreading of glioblastoma remains poorly understood. In this study, we investigated the mechanism underlying intracranial glioblastoma progression. Our findings demonstrate that the mitochondria-derived peptide, humanin, plays a significant role in enhancing glioblastoma progression through the intratumoral activation of the integrin alpha V (ITGAV)–TGF beta (TGFβ) signaling axis. In glioblastoma tissues, humanin showed a significant upregulation in the tumor area compared to the corresponding normal region. Utilizing multiple in vitro pharmacological and genetic approaches, we observed that humanin activates the ITGAV pathway, leading to cellular attachment and filopodia formation. This process aids the subsequent migration and invasion of attached glioblastoma cells through intracellular TGFβR signaling activation. In addition, our in vivo orthotopic glioblastoma model provides further support for the pro-tumoral function of humanin. We observed a correlation between poor survival and aggressive invasiveness in the humanin-treated group, with noticeable tumor protrusions and induced angiogenesis compared to the control. Intriguingly, the in vivo effect of humanin on glioblastoma was significantly reduced by the treatment of TGFBR1 inhibitor. To strengthen these findings, public database analysis revealed a significant association between genes in the ITGAV–TGFβR axis and poor prognosis in glioblastoma patients. These results collectively highlight humanin as a pro-tumoral factor, making it a promising biological target for treating glioblastoma.

IMT030122, A novel engineered EpCAM/CD3/4-1BB tri-specific antibody, enhances T-cell recruitment and demonstrates anti-tumor activity in mouse models of colorectal cancer

Colorectal cancer is a major global health burden, with limited efficacy of traditional treatment modalities in improving survival rates. However, recently advances in immunotherapy has improved treatment outcomes for patients with this cancer. To address the continuing need for improved treatment efficacy, this study introduced a novel tri-specific antibody, IMT030122, that targets EpCAM, 4-1BB, and CD3. We evaluated the pharmacological efficacy and mechanism of action of IMT030122 in vitro and in vivo. In in vitro studies, IMT030122 exhibited differential binding to antigens and cells expressing EpCAM, 4-1BB, and CD3. Moreover, IMT030122 relied on EpCAM-targeted activation of intracellular CD3 and 4-1BB signaling and mediated T cell cytotoxicity specific to HCT116 colorectal cancer cells. In vivo, IMT030122 demonstrated potent anti-tumor activity, significantly inhibiting the growth of colon cancer HCT116 and MC38-hEpCAM subcutaneous grafts. Further pharmacological analysis revealed that IMT030122 recruited lymphocytes from peripheral blood into colorectal cancer tissue and exerted durable anti-tumor activity, predominantly by promoting the activation, proliferation, and differentiation of CD8T cells. Notably, IMT030122 still exhibited anti-tumor efficacy even in the presence of significantly depleted lymphocytes in colorectal cancer tissue. The potent pharmacological activity and anti-tumor effects of IMT030122 suggest it may enhance treatment efficacy and substantially extend the survival of patients with colorectal cancer in the future.

Expression of Concern: “α-Zearalanol attenuates oxLDL-induced ET-1 gene expression, ET-1 secretion and redox-sensitive intracellular signaling activation in human umbilical vein endothelial cells” [Toxicology Letters 179 (3) (10 July 2008) 163–168]

Expression of Concern: “α-Zearalanol attenuates oxLDL-induced ET-1 gene expression, ET-1 secretion and redox-sensitive intracellular signaling activation in human umbilical vein endothelial cells” [Toxicology Letters 179 (3) (10 July 2008) 163–168]

Antiangiogenic therapy: how far is it to upgrade?

AbstractEarly vascular-targeted drugs represented by VEGF single-pathway inhibitors pioneered the idea of regulating the tumor growth microenvironment and enhanced the chemotherapy effect in a variety of tumors. However, their shortcomings of “only winning PFS but not OS” have gradually revealed and warned that drug-resistant growth of tumors is unavoidable after long-term use. The activation of intracellular bypass signaling after inhibiting a single target may be an important reason. In order to eliminate this problem, attempts have been made to expand the combination of drug types and increase the intensity of drug treatment. The possibility of replacing single-target anti-angiogenic therapeutic drugs with multiple targets has also be explored. A new generation of “dual-dimension, multi-pathway” drugs that simultaneously inhibit multiple targets both in tumors and microvascular endothelial cells can be used either as single drugs in multiple tumor types, or in combination with chemotherapy, EGFR-TKI, or even immunological drugs. It demonstrates good efficacy and great potential and hope for upgrading vascular targeted therapy. It also enlightens us that future treatment and new drug development strategies cannot rely solely on the most precise target inhibition, but should have a “dual-dimension, multi-pathway” integration concept that looks at the overall situation from one corner.

A Review of Approaches to Potentiate the Activity of Temozolomide against Glioblastoma to Overcome Resistance.

A glioblastoma (GBM) is one of the most aggressive, infiltrative, and treatment-resistant malignancies of the central nervous system (CNS). The current standard of care for GBMs include maximally safe tumor resection, followed by concurrent adjuvant radiation treatment and chemotherapy with the DNA alkylating agent temozolomide (TMZ), which was approved by the FDA in 2005 based on a marginal increase (~2 months) in overall survival (OS) levels. This treatment approach, while initially successful in containing and treating GBM, almost invariably fails to prevent tumor recurrence. In addition to the limited therapeutic benefit, TMZ also causes debilitating adverse events (AEs) that significantly impact the quality of life of GBM patients. Some of the most common AEs include hematologic (e.g., thrombocytopenia, neutropenia, anemia) and non-hematologic (e.g., nausea, vomiting, constipation, dizziness) toxicities. Recurrent GBMs are often resistant to TMZ and other DNA-damaging agents. Thus, there is an urgent need to devise strategies to potentiate TMZ activity, to overcome drug resistance, and to reduce dose-dependent AEs. Here, we analyze major mechanisms of the TMZ resistance-mediated intracellular signaling activation of DNA repair pathways and the overexpression of drug transporters. We review some of the approaches investigated to counteract these mechanisms of resistance to TMZ, including the use of chemosensitizers and drug delivery strategies to enhance tumoral drug exposure.

The role of extracellular vesicle fusion with target cells in triggering systemic inflammation

Extracellular vesicles (EVs) play a crucial role in intercellular communication by transferring bioactive molecules from donor to recipient cells. As a result, EV fusion leads to the modulation of cellular functions and has an impact on both physiological and pathological processes in the recipient cell. This study explores the impact of EV fusion on cellular responses to inflammatory signaling. Our findings reveal that fusion renders non-responsive cells susceptible to inflammatory signaling, as evidenced by increased NF-κB activation and the release of inflammatory mediators. Syntaxin-binding protein 1 is essential for the merge and activation of intracellular signaling. Subsequent analysis show that EVs transfer their functionally active receptors to target cells, making them prone to an otherwise unresponsive state. EVs in complex with their agonist, require no further stimulation of the target cells to trigger mobilization of NF-κB. While receptor antagonists were unable to inhibit NF-κB activation, blocking of the fusion between EVs and their target cells with heparin mitigated inflammation in mice challenged with EVs.

Intranasal insulin attenuates hypoxia-ischemia-induced short-term sensorimotor behavioral disturbances, neuronal apoptosis, and brain damage in neonatal rats

There is a significant need for additional therapy to improve outcomes for newborns with acute Hypoxic-ischemic (HI) encephalopathy (HIE). New evidence suggests that insulin could be neuroprotective. This study aimed to investigate whether intranasal insulin attenuates HI-induced brain damage and neurobehavioral dysfunction in neonatal rats. Postnatal day 10 (P10), Sprague-Dawley rat pups were randomly divided into Sham + Vehicle, Sham + Insulin, HI + Vehicle, and HI + Insulin groups with equal male-to-female ratios. Pups either had HI by permanent ligation of the right common carotid artery followed by 90 min of hypoxia (8% O2) or sham surgery followed by room air exposure. Immediately after HI or Sham, pups were given fluorescence-tagged insulin (Alex-546-insulin)/vehicle, human insulin (25 μg), or vehicle in each nare under anesthesia. Shortly after administration, widespread Alex-546-insulin-binding cells were detected in the brain, primarily co-localized with neuronal nuclei-positive neurons on double-immunostaining. In the hippocampus, phospho-Akt was activated in a subset of Alex-546-insulin double-labeled cells, suggesting activation of the Akt/PI3K pathway in these neurons. Intranasal insulin (InInsulin) reduced HI-induced sensorimotor behavioral disturbances at P11. InInsulin prevented HI-induced increased Fluoro-Jade C+ degenerated neurons, cleaved caspase 3+ neurons, and volume loss in the ipsilateral brain at P11. There was no sex-specific response to HI or insulin. The findings confirm that intranasal insulin provides neuroprotection against HI brain injury in P10 rats associated with activation of intracellular cell survival signaling. If further pre-clinical research shows long-term benefits, intranasal insulin has the potential to be a promising non-invasive therapy to improve outcomes for newborns with HIE.

Dimerization of melanocortin 4 receptor controls puberty onset and body size polymorphism.

Xiphophorus fish exhibit a clear phenotypic polymorphism in puberty onset and reproductive strategies of males. In X. nigrensis and X. multilineatus, puberty onset is genetically determined and linked to a melanocortin 4 receptor (Mc4r) polymorphism of wild-type and mutant alleles on the sex chromosomes. We hypothesized that Mc4r mutant alleles act on wild-type alleles by a dominant negative effect through receptor dimerization, leading to differential intracellular signaling and effector gene activation. Depending on signaling strength, the onset of puberty either occurs early or is delayed. Here, we show by Förster Resonance Energy Transfer (FRET) that wild-type Xiphophorus Mc4r monomers can form homodimers, but also heterodimers with mutant receptors resulting in compromised signaling which explains the reduced Mc4r signaling in large males. Thus, hetero- vs. homo- dimerization seems to be the key molecular mechanism for the polymorphism in puberty onset and body size in male fish.

Nutritional Value of Yogurt as a Protein Source: Digestibility/Absorbability and Effects on Skeletal Muscle.

Yogurt is a traditional fermented food that is accepted worldwide for its high palatability and various health values. The milk protein contained in yogurt exhibits different physical and biological properties from those of non-fermented milk protein due to the fermentation and manufacturing processes. These differences are suggested to affect the time it takes to digest and absorb milk protein, which in turn will influence the blood levels of amino acids and/or hormones, such as insulin, and thereby, the rate of skeletal muscle protein synthesis via the activation of intracellular signaling, such as the mTORC1 pathway. In addition, based on the relationship between gut microbiota and skeletal muscle conditions, yogurt, including lactic acid bacteria and its metabolites, has been evaluated for its role as a protein source. However, the substantial value of yogurt as a protein source and the additional health benefits on skeletal muscle are not fully understood. The purpose of this review is to summarize the research to date on the digestion and absorption characteristics of yogurt protein, its effect on skeletal muscle, and the contribution of lactic acid bacterial fermentation to these effects.

The D647N mutation of FGFR1 induces ligand-independent receptor activation

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

Re-routing GPR56 signalling using Gα12/13 G protein chimeras.

Adhesion G protein-coupled receptors (aGPCRs) constitute the second largest subclass of the GPCR superfamily. Although canonical GPCRs are explored pharmacologically as drug targets, no clinically approved drugs target the aGPCR family so far. The aGPCR GPR56/ADGRG1 stands out as an especially promising target, given its direct link to the monogenetic disease bilateral frontoparietal polymicrogyria and implications in cancers. Key to understanding GPCR pharmacology has been mapping out intracellular signalling activity. Detection of GPCR signalling in the Gαs /Gαi /Gαq G protein pathways is feasible with second messenger detection systems. However, in the case of Gα12/13 -coupled receptors, like GPR56, signalling detection is more challenging due to the lack of direct second messenger generation. To overcome this challenge, we engineered a Gαq chimera to translate Gα12/13 signalling. We show the ability of the chimeric GαΔ6q12myr and GαΔ6q13myr to translate basal Gα12/13 signalling of GPR56 to a Gαq readout in transcription factor luciferase reporter systems and show that the established peptide ligands (P7 and P19) function to enhance this signal. We further demonstrate the ability to directly influence the generation of second messengers in inositol-3-phosphate assays. In the future, these chimeric G proteins could facilitate basic functional studies, drug screenings and deorphanization of other aGPCRs.

Diverse effects of prostacyclin on angiogenesis-related processes in the porcine endometrium

Angiogenesis is important for endometrial remodeling in mature females. The endometrium synthesizes high amounts of prostacyclin (PGI2) but the role of PGI2 in angiogenesis-related events in this tissue was not fully described. In the present study, porcine endometrial endothelial (pEETH) cells and/or a swine umbilical vein endothelial cell line (G1410 cells) were used to determine the regulation of PGI2 synthesis and PGI2 receptor (PTGIR) expression by cytokines and to evaluate the effect of PGI2 on pro-angiogenic gene expression, intracellular signaling activation, cell proliferation and migration, cell cycle distribution, and capillary-like structure formation. We found that IL1β, IFNγ, and/or TNFα increased PGI2 secretion and PTGIR expression in pEETH cells. Iloprost (a PGI2 analogue) acting through PTGIR enhanced the transcript abundance of KDR, FGFR2, and ANGPT2 and increased proliferation of pEETH cells. This latter was mediated by PI3K and mTOR activation. In support, transfection of G1410 cells with siRNA targeting PGI2 synthase decreased pro-angiogenic gene expression and cell proliferation. Furthermore, iloprost accelerated the gap closure and promoted cell cycle progression. Intriguingly, the formation of capillary-like structures was inhibited but not completely blocked by iloprost. These findings point to a complex pleiotropic role of PGI2 in angiogenesis-related events in the porcine uterus.

Cardiotoxicity is mediated by the NLRP3 inflammasome in hiPSC-derived endothelial cells from patients with immune checkpoint inhibitor-related myocarditis

In association with immune checkpoint inhibitors (ICI) anticancer therapy, myocarditis (ICI-M), have emerged with a mortality rate reaching 50%. The IFN-γ pathway and inflammasome regulatory proteins seem to be specifically involved in ICI-M, but the mechanism of action remains unclear. In the cardiovascular system, the endocardial and vascular endothelium is involved in the regulation of the contractile function of cardiomyocytes. Our hypothesis is that the cardiotoxicity induced by the infiltration of immune cells into the myocardium is partly due to an exacerbation of inflammasome activation in endothelial cells, which subsequently affects the cardiomyocytes function. Our aim is to characterize the role of endothelial cells in ICI-related myocarditis in a unique model of induced pluripotent stem cells (hiPSC)-derived cardiac cells. The hiPSC were generated from melanoma patients who received anti-PD-1/anti-CTLA-4 immunotherapy and followed at the Cardio-Oncology Centre (North Hospital, Marseille). Twelve hiPSC clones were generated from patients who developed myocarditis (ICI-M, 6 clones from 3 patients), and patients who did not develop cardiotoxicity (non-ICI-M, 6 clones from 3 patients). hiPSC-derived endothelial cells (hiPSC-EC) were differentiated from hiPSC clones and stimulated by 10 ng/mL of IFN-γ during 48 h. IFN-γ induced a decrease in cell viability, as well as an increase in the expression of transcripts of genes associated with the JAK/STAT and NLRP3 inflammasome pathways in hiPSC-EC. However, the decrease in cell viability was significantly greater in hiPSC-EC from the ICI-M group compared to the non-ICI-M group. IFN-γ activation of inflammatory intracellular signalling is greater in the ICI-M group, in particular overexpression of the jak1, jak2, stat3 and gbp6 transcripts. At the protein level, in this ICI-M group, the IFN-γ-induced increase of MHC-I and PD-L1 is greater than in the non-ICI-M group. IFN-γ stimulation revealed specific regulations in endothelial cells derived from hiPSC clones from patients with ICI-related myocarditis. We are now analysing the apoptotic and inflammatory activity of hiPSC-EC by real-time microscopy monitoring of caspases (Incucyte S3) and by assaying pro-inflammatory molecules in the supernatants (ELISA). This study will allow to establish the role played by endothelial cells in cardiac toxicity induced by ICI immunotherapy.

Calmodulin Regulates Transient Receptor Potential TRPM3 and TRPM8-Induced Gene Transcription.

Calmodulin is a small protein that binds Ca2+ ions via four EF-hand motifs. The Ca2+/calmodulin complex as well as Ca2+-free calmodulin regulate the activities of numerous enzymes and ion channels. Here, we used genetic and pharmacological tools to study the functional role of calmodulin in regulating signal transduction of TRPM3 and TRPM8 channels. Both TRPM3 and TRPM8 are important regulators of thermosensation. Gene transcription triggered by stimulation of TRPM3 or TRPM8 channels was significantly impaired in cells expressing a calmodulin mutant with mutations in all four EF-hand Ca2+ binding motifs. Similarly, incubation of cells with the calmodulin inhibitor ophiobolin A reduced TRPM3 and TRPM8-induced signaling. The Ca2+/calmodulin-dependent protein phosphatase calcineurin was shown to negatively regulate TRPM3-induced gene transcription. Here, we show that TRPM8-induced transcription is also regulated by calcineurin. We propose that calmodulin plays a dual role in regulating TRPM3 and TRPM8 functions. Calmodulin is required for the activation of TRPM3 and TRPM8-induced intracellular signaling, most likely through a direct interaction with the channels. Ca2+ influx through TRPM3 and TRPM8 feeds back to TRPM3 and TRPM8-induced signaling by activation of the calmodulin-regulated enzyme calcineurin, which acts as a negative feedback loop for both TRPM3 and TRPM8 channel signaling.

The emerging role of estrogen's non-nuclear signaling in the cardiovascular disease.

Sexual dimorphism exists in the epidemiology of cardiovascular disease (CVD), which indicates the involvement of sexual hormones in the pathophysiology of CVD. In particular, ample evidence has demonstrated estrogen's protective effect on the cardiovascular system. While estrogen receptors, bound to estrogen, act as a transcription factor which regulates gene expressions by binding to the specific DNA sequence, a subpopulation of estrogen receptors localized at the plasma membrane induces activation of intracellular signaling, called "non-nuclear signaling" or "membrane-initiated steroid signaling of estrogen". Although the precise molecular mechanism of non-nuclear signaling as well as its physiological impact was unclear for a long time, recent development of genetically modified animal models and pathway-selective estrogen receptor stimulant bring new insights into this pathway. We review the published experimental studies on non-nuclear signaling of estrogen, and summarize its role in cardiovascular system, especially focusing on: (1) the molecular mechanism of non-nuclear signaling; (2) the design of genetically modified animals and pathway-selective stimulant of estrogen receptor.

RAGE Inhibitors in Neurodegenerative Diseases.

Nonenzymatic reactions of reducing sugars with primary amino groups of amino acids, proteins, and nucleic acids, followed by oxidative degradations would lead to the formation of advanced glycation endproducts (AGEs). The AGEs exert multifactorial effects on cell damage leading to the onset of neurological disorders. The interaction of AGEs with the receptors for advanced glycation endproducts (RAGE) contribute to the activation of intracellular signaling and the expression of the pro-inflammatory transcription factors and various inflammatory cytokines. This inflammatory signaling cascade is associated with various neurological diseases, including Alzheimer's disease (AD), secondary effects of traumatic brain injury (TBI), amyotrophic lateral sclerosis (ALS), and diabetic neuropathy, and other AGE-related diseases, including diabetes and atherosclerosis. Furthermore, the imbalance of gut microbiota and intestinal inflammation are also associated with endothelial dysfunction, disrupted blood-brain barrier (BBB) and thereby the onset and progression of AD and other neurological diseases. AGEs and RAGE play an important role in altering the gut microbiota composition and thereby increase the gut permeability and affect the modulation of the immune-related cytokines. The inhibition of the AGE-RAGE interactions, through small molecule-based therapeutics, prevents the inflammatory cascade of events associated with AGE-RAGE interactions, and thereby attenuates the disease progression. Some of the RAGE antagonists, such as Azeliragon, are currently in clinical development for treating neurological diseases, including AD, although currently there have been no FDA-approved therapeutics based on the RAGE antagonists. This review outlines the AGE-RAGE interactions as a leading cause of the onset of neurological diseases and the current efforts on developing therapeutics for neurological diseases based on the RAGE antagonists.

Abstract 5744: Decreasing toxicity of immunocytokines by transient and selective inhibition of their intracellular signaling activation

Abstract The use of cytokine-based therapeutics has traditionally been limited by systemic toxicity, but it is now becoming evident that it may be possible to circumvent this problem in a number of ways. Tumor-targeting antibody-cytokine fusion proteins (also called “immunocytokines”) typically allow dose escalation to therapeutically active regimens as a result of their selective localization at the site of disease (Neri 2019; Helguera, Morrison, and Penichet 2002). Despite the enhancement of the therapeutic index, when the concentration of the payload in blood exceeds a certain threshold, patients may experience dose-limiting immune-related adverse events (irAEs). In most cases, irAEs disappear when the immunocytokine is cleared from circulation (Ko et al. 2004; Spitaleri et al. 2013; Rudman et al. 2011). Here, we describe an innovative approach to mask off-target activity of immunocytokines by transient inhibition of their intracellular signaling cascade. Small molecules are particularly attractive for this application since their serum half-life matches the clearance rate of the immunocytokine from circulation. L19-TNF and L19-IL12 are two clinical-stage antibody-cytokine fusion proteins that display potent activity by triggering the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and Janus Kinase 2 (JAK2) signaling pathways, respectively (Amin et al. 2018; Hu et al. 2021). In a first study, GSK’963, a potent small molecule inhibitor of RIPK1, was tested in tumor-bearing mice with the aim to reduce acute systemic toxicity associated with TNF signaling. Transient inhibition of RIPK1 allowed the administration of L19-murTNF doses which would otherwise be lethal. The combination with GSK’963 did not affect the selective localization of the immunocytokine to tumors. Moreover, L19-murTNF was still able to induce tumor necrosis and to exert potent anti-cancer activity. In a second study, tumor-bearing mice receiving L19-murIL12 were pretreated with Ruxolitinib, a commercially available JAK2 inhibitor. The addition of Ruxolitinib could significantly improve the tolerability profile of L19-murIL12 without affecting the anti-cancer properties of the immunocytokine. In clinical trials, it has been observed that IL12 treatment can induce cytokine release syndrome and liver damage (Atkins et al. 1997). Our data demonstrate that the pre-treatment with Ruxolitinib restricted the pro-inflammatory effects of L19-murIL12 to the tumor site, protecting the mice from body weight loss as well as reducing blood cytokine levels and hepatotoxicity. Overall, this preclinical work is of clinical relevance, as patients treated with targeted cytokines could potentially benefit from judicious combinations treatments using small molecule inhibitors. Citation Format: Giulia Rotta, Ettore Gilardoni, Domenico Ravazza, Jacqueline Mock, Samuele Cazzamalli, Roberto De Luca, Emanuele Puca, Dario Neri, Sheila Dakhel Plaza. Decreasing toxicity of immunocytokines by transient and selective inhibition of their intracellular signaling activation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5744.

Abstract 6416: Statins inhibit onco-dimerization of 4Ig-B7-H3

Abstract B7-H3 (CD276) is a member of the B7 family of immune regulators, but unlike the immune checkpoint targets that have revolutionized cancer therapy, blockade has resulted in mixed outcomes. B7-H3 has 2Ig and 4Ig isoforms, has no known direct ligand, and many of the physiological functions remain elusive. While protein expression of B7-H3 is differentially expressed on many solid tumors and correlates with poor survival, mechanisms of how B7-H3 elicits a pro-tumorigenic phenotype are still poorly understood, significantly hampering therapeutic efforts for targeting. Using a facile, recombinase-enhanced split-luciferase reporter, we visualized 4Ig-B7-H3 homodimerization in live U2OS cells by bioluminescence imaging and confirmed 4Ig-B7-H3 dimerization using a second non-invasive imaging technique: fluorescence lifetime imaging microscopy (FLIM) where 4Ig-B7-H3 was exogenously re-expressed following knockout of CD276 in HeLa cervical cancer cells. Homodimerization of 4Ig-B7-H3 correlated with enhanced clonogenic cell growth in HeLa, SKOv3, MDA-MB-231, and U2OS cells and tumor growth in vivo for several models. This phenotype was reversed following knockout of B7-H3 and rescued upon lentiviral re-expression. By RPPA and phospho-kinase array analysis, increases in PI3K/AKT signaling, Jak/Stat pathway, and modulators of HIF1α and NF-κβ pathways were observed with B7-H3 expression and homodimerization (p<0.05), all which are known to support increased oncogenic functions, such as increased glycolysis, increased survival and proliferation, as well as cytokine modulation and immune evasion. Next, capitalizing on our bioluminescence-based reporter of 4Ig-B7-H3 homodimerization, we performed a high-throughput small molecule screen with a 5,362 Bioactive compound library to identify modulators of 4Ig-B7-H3 dimerization. Notably, our HTS identified several HMG-CoA reductase inhibitors (statins) as significant inhibitors of B7-H3 dimerization (p<0.01). Treatment with 1 μM mevastatin or atorvastatin calcium inhibited clonogenic growth of HeLa cervical, SKOv3 ovarian and MDA-MB-231 breast cancer cells, which all endogenously express B7-H3. In vivo, treatment with 10 mg/kg of atorvastatin calcium reduced tumor growth and increased mouse survival (p=0.035) of SKOv3-ip tumors and produced functional cures (15-30%) of HeLa or MDA-MB-231 xenografts in nude mice in a B7-H3 tumor expression-dependent manner, proven through knockout and rescue experiments in live cells and animals. Thus, in the context of studies to dissect the tumor-intrinsic mechanism(s) of B7-H3 biochemical functions and activation, we identified a novel, dimerization-dependent oncogenic role for 4Ig-B7-H3 that increased oncogenic intracellular signaling activation and was modulated by statin treatment. This may provide a molecular mechanism for the statin-mediated immune modulation and enhanced cancer survival clinically reported for several tumor types. Citation Format: Margie Nicole Sutton, Sarah E. Glazer, Riccardo Muzzioli, Ping Yang, Seth T. Gammon, David Piwnica-Worms. Statins inhibit onco-dimerization of 4Ig-B7-H3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6416.

A Candidate Gliotransmitter, L-β-Aminoisobutyrate, Contributes to Weight Gain and Metabolic Complication Induced by Atypical Antipsychotics.

Lurasidone and quetiapine are effective atypical mood-stabilizing antipsychotics, but lurasidone and quetiapine are listed as lower-risk and high-risk for weight gain/metabolic complications, respectively. The pathophysiology of the discrepancy of metabolic adverse reactions between these antipsychotics remains to be clarified. The GABA isomer, β-aminoisobutyric acid (BAIBA) enantiomer, was recently re-discovered as myokine via an AMP-activated protein kinase activator (AMPK) enhancer and inhibitory gliotransmitter. Notably, activation of AMPK in peripheral organs improves, but in the hypothalamus, it aggravates metabolic disturbances. Therefore, we determined effects of chronic administration of lurasidone and quetiapine on intracellular and extracellular levels of the BAIBA enantiomer. L-BAIBA is a major BAIBA enantiomer in the hypothalamus and astrocytes, whereas L-BAIBA only accounted for about 5% of total plasma BAIBA enantiomers. Chronic lurasidone administration did not affect body weight but decreased the L-BAIBA level in hypothalamus and cultured astrocytes, whereas chronic quetiapine administration increased body weight and the L-BAIBA level in hypothalamus and astrocytes. Contrary, neither lurasidone nor quetiapine affected total plasma levels of the BAIBA enantiomer since D-BAIBA levels were not affected by these antipsychotics. These results suggest that activation of intracellular L-BAIBA signaling is, at least partially, involved in the pathophysiology of metabolic adverse reaction of quetiapine. Furthermore, this study also demonstrated that lurasidone and quetiapine suppressed and enhanced astroglial L-BAIBA release induced by ripple-burst stimulation (which physiologically contributes to cognitive memory integration during sleep), respectively. Therefore, L-BAIBA probably contributes to the pathophysiology of not only metabolic adverse reactions, but also a part of clinical action of lurasidone or quetiapine.

Mechanisms of action of extracellular miRNAs

The mechanisms of action of extracellular miRNAs are given in the scientific review. To write the article, information was searched using Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library databases. The authors state that miRNAs are generated by producer cells and then packaged in extracellular vesicles and delivered to other cells by functionally active structures. Extracellular vesicles transport miRNA in a paracrine and endocrine manner. It is emphasized that the mechanism of action of extracellular microRNAs depends on their association with extracellular vesicles. MicroRNAs associated with extracellular vesicles are taken up by cells and act as intracellular microRNAs. MicroRNAs associated and not associated with extracellular vesicles, by interacting with specific receptors, induce the activation of intracellular signaling, most often pro-inflammatory pathways. Extracellular vesicles interact with the membrane of the recipient cell through their surface proteins in a ligand-receptor manner. It is shown that specific proteins of extracellular vesicles, such as major histocompatibility complex class I and class II proteins, transferrin receptors and tetraspanins, induce disruption of some signaling pathways of target cells. Scientists believe that exosomes and ectosomes are absorbed by recipient cells using clathrin-mediated endocytosis, phagocytosis, and/or macropinocytosis. After delivery to target cells, miRNAs regulate the translation of their target genes. Extracellular vesicles circulating in the blood serum of healthy people are believed to have antifibrogenic effects and contain miRNAs that inhibit the activity of hematopoietic stem cells or damaged hepatocytes. Scientists believe that receptor-independent penetration of the hepatitis C virus into hepatocytes can be carried out with the help of exosomes. Exosomal miRNAs and miRNAs not associated with exosomes are able to bind to certain cellular receptors and activate intracellular signaling pathways associated with them.