Direct Crosstalk Research Articles

Implications of glioblastoma-derived exosomes in modifying the immune system: state-of-the-art and challenges.

Glioblastoma is a brain cancer with a poor prognosis. Failure of classical chemotherapy and surgical treatments indicates that new therapeutic approaches are needed. Among cell-free options, exosomes are versatile extracellular vesicles (EVs) that carry important cargo across barriers suchas the blood-brain barrier (BBB) to their target cells. Thismakes exosomes an interesting option for the treatment ofglioblastoma. Moreover, exosomes can comprise many therapeutic cargos, including lipids, proteins, and nucleic acids,sampled from special intercellular compartments of their origin cell. Cells exposed to various immunomodulatory stimulican generate exosomes enriched in specific therapeutic molecules. Notably, the secretion of exosomes could modify theimmune response in innate and adaptive immune systems. For instance, glioblastoma-associated exosomes (GBex) uptake bymacrophages could influence macrophage dynamics (e.g., shifting CD markers expression). Expression of critical immunoregulatory proteins such as cytotoxic T-lymphocyte antigen-1 (CTLA1) and programmed death-1 (PD-1) on GBex indicates the direct crosstalk of these nano-size vesicles with the immune system. The present study reviews the role of exosomes in immune system cells, including Bcells, Tcells, natural killer (NK) cells, and dendritic cells (DCs), as well as novel technologies in the field.

Pericytes Modulate Third-Generation Tyrosine Kinase Inhibitor Sensitivity in EGFR-Mutated Lung Cancer Cells Through IL32-β5-Integrin Paracrine Signaling.

EGFR-mutated lung cancer patients sometimes display restricted responses to third-generation tyrosine kinase inhibitors (TKIs), potentially attributable to undervalued input from stromal cells, notably pericytes (PCs). The study shows that PCs isolated from EGFR-mutated patients have a unique secretome profile, notably secreting IL32 and affecting signaling pathways and biological processes linked to TKI sensitivity. Clinical evidence, supported by single-cell RNA sequencing and multiplex immunostaining of tumor tissues, confirms the presence of IL32-expressing pericytes closely interacting with β5-integrin-expressing cancer cells in EGFR-mutated patients, impacting therapeutic response and prognosis. Co-culture and conditioned medium experiments demonstrate that PCs reduce TKI effectiveness in EGFR-mutated cancer cells, a reversible phenomenon through silencing IL32 expression in PCs or depleting the IL32 receptor β5-integrin on cancer cells, thereby restoring cancer cell sensitivity. Mechanistically, it is shown that YY1 signaling upregulates IL32 secretion in PCs, subsequently activating the β5-integrin-Src-Akt pathway in EGFR-mutated cancer cells, contributing to their TKI sensitivity. In animal studies, co-injection of cancer cells with PCs compromises TKI effectiveness, independently of blood vessel functions, while inhibition of β5-integrin restores tumor cell sensitivity. Overall, the findings highlight direct crosstalk between cancer cells and pericytes, impacting TKI sensitivity via IL32-β5-integrin paracrine signaling, proposing an enhanced therapeutic approach for EGFR-mutated patients.

A Numerical Assessment of the Effect of Concatenating Arbitrary Uncoupled Multicore Fiber Segments on Intercore Crosstalk in Long-Haul Communication Links

Random core dependent loss (CDL) has been shown to increase the direct average intercore crosstalk (ICXT) power in long-haul uncoupled multicore fiber (MCF) links. Longer links are composed of multiple MCF segments, and random CDL may arise on these links from manufacturing imperfections. During link implementation, other random effects may arise and enhance the ICXT power. In this work, the effect of concatenating MCF segments with random characteristics on the direct average ICXT power in long-haul links is assessed numerically by studying the influence of the randomness of segment length, coupling coefficient, and random CDL on the mean, standard deviation, relative spread, and excess kurtosis of the ICXT power. The numerical results show that the segment length randomness marginally affects the ICXT power. For 2000 km long links and a 6 dB maximum random variation of the coupling coefficients, the mean almost doubles and the standard deviation almost triples, relative to considering only random CDL. However, the effect of the coupling coefficients randomness on the relative spread and excess kurtosis is reduced, not affecting significantly the nearly Gaussian distribution of the direct average ICXT power and the excess of direct average ICXT power (less than a 0.26 dB increase relative to considering only random CDL).

Interaction of acoustic waves with spin waves using a GHz operating GaN/Si SAW device with a Ni/NiFeSi layer between its IDTs.

A two port surface acoustic wave (SAW) device was developed to be used for the control and excitation via spin waves (SW). The structure was manufactured using advanced nanolithography techniques, on GaN/Si, enabling fundamental Rayleigh interdigitated transducer (IDT) resonances in GHz frequency range. The ferromagnetic resonance of the magnetostrictive Ni/NiFeSi layer placed between the IDTs of the SAW device can be tuned to the SAW resonance frequency by magnetic fields. Using structures with finger and interdigit spacing of 170 nm and 100 nm, fundamental Rayleigh IDT resonance frequencies of 6.4 and 10.4 GHz have been obtained. Coupling of SAW to SW was demonstrated through transmission measurements at the fundamental Rayleigh frequencies in a magnetic field, μ0H from -280 to +280 mT, at different angles (θ) between the SAW propagation direction and the magnetic field direction. For the 6.4 GHz resonator a maximum decrease of about 1.2 dB occurred in |S21|, at μ0H = 30 mT and at θ = 45. Time-gated processing of the frequency domain raw data was used to remove the direct electromagnetic cross talk and triple transit effects. Nonreciprocity associated to the coupling was analyzed for the two SAW structures. The quantitative influence of the magnetic field strength on the phase of the transmission parameters is also presented.

Statistical characterization of the effect of random core loss on the intercore crosstalk in long-haul uncoupled multicore fiber links.

We characterize the statistical properties of direct average intercore crosstalk (ICXT) in long-haul uncoupled multicore fiber (MCF) links consisting of concatenated MCF segments, where core dependent loss (CDL) in each segment varies randomly. Numerical simulation results show that the direct average ICXT power at the output of long-haul MCF links with random CDL is well described by a Gaussian distribution. A statistical distribution, which accounts for only two values of core loss (the highest and lowest core loss) with equal probability, is proposed as the worst-case distribution of core loss resulting in the maximum excess of direct average ICXT power. With this distribution, practical values of random CDL, 20 MCF segments per span and 2000 km-long links, analytical and simulation results show the maximum excess of direct average ICXT power does not exceed 0.25 dB, being further lower for a higher number of segments. An analytical approximation for the maximum excess of direct average ICXT power in long-haul uncoupled MCF links with similar spans is presented. Demonstrating high accuracy (with less than 0.02 dB discrepancy relative to simulation results in all cases evaluated), this approximation provides a simple and efficient means for estimating the worst-case impact of random CDL on ICXT power in long-haul MCF links.

FGD5 in basal cells induces CXCL14 secretion that initiates a feedback loop to promote murine mammary epithelial growth and differentiation

The interactions of environmental compartments with epithelial cells are essential for mammary gland development and homeostasis. Currently, the direct crosstalk between the endothelial niche and mammary epithelial cells remains poorly understood. Here, we show that faciogenital dysplasia 5 (FGD5) is enriched in mammary basal cells (BCs) and mediates critical interactions between basal and endothelial cells (ECs) in the mammary gland. Conditional deletion of Fgd5 reduced, whereas conditional knockin of Fgd5 increased, the engraftment and expansion of BCs, regulating ductal morphogenesis in the mammary gland. Mechanistically, murine mammary BC-expressed FGD5 inhibited the transcriptional activity of activating transcription factor 3 (ATF3), leading to subsequent transcriptional activation and secretion of CXCL14. Furthermore, activation of CXCL14/CXCR4/ERK signaling in primary murine mammary stromal ECs enhanced the expression of HIF-1α-regulated hedgehog ligands, which initiated a positive feedback loop to promote the function of BCs. Collectively, these findings identify functionally important interactions between BCs and the endothelial niche that occur through the FGD5/CXCL14/hedgehog axis.

TGFβR1 inhibition drives hepatocellular carcinoma proliferation through induction of toll-like-receptor signalling.

Transforming growth factor (TGF)-β and toll-like receptors (TLRs) have been shown to independently modulate the proliferation of hepatocellular carcinoma (HCC). Since a direct cross-talk between these two signalling pathways in HCC has not been clearly described before, we aimed here to explore the possibility of such interaction. A human HCC tissue array (n = 20 vs. four control samples), human HCC samples (n = 10) and steatohepatitis-driven murine HCC samples (control, NASH and HCC; n = 6/group) were immunostained for TGFβR1, pSMAD2, TRAF6, IRAK1 and PCNA. The results were confirmed by immunoblotting. Effects of constant activation of the SMAD pathway by constitutive expression of ALK5 or knockdown of mediators of TLR signalling, IRAK1 and MyD88, on HCC proliferation, were investigated in the HCC cell line (HUH-7) after treatment with TGFβ1 cytokine or TGFβR1 kinase inhibitor (LY2157299) using PCNA and MTS assay. TGFβR1 expression is decreased in human and murine HCC and associated with downregulated pSMAD2, but increased IRAK1, TRAF6 and PCNA staining. TGFβR1 kinase inhibition abolished the cytostatic effects of TGFβ1 and led to the induction of IRAK1, pIRAK1 and elevated mRNA levels of TLR-9. Overexpression of ALK5 and knockdown of MyD88 or IRAK1 augmented the cytostatic effects of TGFβ1 on HUH-7. In another epithelial HCC cell line, that is, HepG2, TGFβR1 kinase inhibitor similarly elevated cellular proliferation. There is a balance between the canonical SMAD-driven tumour-suppressing arm and the non-canonical tumour-promoting arm of TGFβ signalling. Disruption of this balance, by inhibition of the canonical pathway, induces HCC proliferation through TLR signalling.

Abstract PR09: Pancreatic cancer cachexia is mediated by tumor-derived PTHrP

Abstract Purpose: Our prior work has established that metastasis is initiated by PTHrP-driven mechanisms in PDAC. PTHLH (the gene encoding the PTHrP protein) is directly adjacent to and co-amplified along with KRAS, and this amplicon is a marker of squamous/quasi-mesenchymal/basal-like PDAC subtypes. Thus, PTHrP marks highly aggressive subsets of PDAC patients. We have generated KPCY-PthlhLoxP (herein KPCY-PthrpcKO) mice and observed a near doubling of overall survival relative to KPCY controls. Intriguingly, recent evidence has emerged for PTHrP’s role in cachexia-associated adipose tissue wasting and we posit that the dramatic survival extension in KPCY-PthrpcKO mice may be due to reduced cachexia. Results: In PDAC patients, PTHrP is co-amplified along with KRAS and correlates with significantly decreased overall survival. We generated KPCY-PthrpcKO mice and showed that they have reduced tumor burden and dramatically increased overall survival relative to KPCY controls. In parallel experiments, we treated mice with an anti-PTHrP neutralizing monoclonal antibody, which similarly reduced tumor growth and extended survival. Upon further analysis, we observed that the overall body condition of KPCY-PthrpcKO mice (and anti-PTHrP treated KPCY mice) was greatly improved, with less loss of adipose and muscle tissue. Mechanistic studies revealed that tumor cell-derived PTHrP signaling to adipocytes in white adipose tissue depots mediates cachexia. Specifically, we found that adipose tissue wasting and lipolysis were greatly reduced upon deletion or pharmacological inhibition of PTHrP. In the same vein, we uncovered that PTHrP is driving an adipocyte “browning” process, a phenomenon where energy-storing white adipocytes transdifferentiate into an energy-expending brown adipocyte-like state (often referred to as “beige” adipocytes). Preliminary results indicate direct tumor cell-adipocyte crosstalk with tumor cell secreted PTHrP binding to its cognate receptor, PTH1R, on adipocytes and activating a thermogenic (heat generating) gene program, likely through a PTH1R-PKA-CREB1 signaling axis. We additionally found that muscle wasting was reduced in KPCY-PthrpcKO mice and anti-PTHrP neutralizing antibody treated KPCY mice. Thus, genetic deletion and pharmacological inhibition of PTHrP in vivo led to a profound reduction in cachexia-related adipose tissue wasting and muscle atrophy. Re-introduction of PTHrP into a PDAC cell line with low cachexia-inducing potential (and low baseline PTHrP) dramatically increased the degree of cachexia observed upon orthotopic implantation. Additionally, mice implanted with PTHrP overexpressing tumor cells had a reduction in overall survival along with decreased overall body condition. Therefore, PTHrP is both necessary and sufficient to induce cachexia. Conclusions: This work has demonstrated the importance of the previously unappreciated roles of PTHrP signaling in driving pancreatic cancer cachexia and adipose tissue browning, and future studies will look to translate anti-PTHrP therapy into clinical trials. Citation Format: Yamini Ogoti, Jessica Peura, Calvin Johnson, Ekaterina Korobkina, Maximilian Wengyn, Robert J. Norgard, Richard Kremer, David A. Guertin, Jason R. Pitarresi. Pancreatic cancer cachexia is mediated by tumor-derived PTHrP [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr PR09.

N-acetyltransferase NAT10 controls cell fates via connecting mRNA cytidine acetylation to chromatin signaling.

Cell fate transition involves dynamic changes of gene regulatory network and chromatin landscape, requiring multiple levels of regulation, yet the cross-talk between epitranscriptomic modification and chromatin signaling remains largely unknown. Here, we uncover that suppression of N-acetyltransferase 10 (NAT10), the writer for mRNA N4-acetylcytidine (ac4C) modification, can notably affect human embryonic stem cell (hESC) lineage differentiation and pluripotent reprogramming. With integrative analysis, we identify that NAT10-mediated ac4C modification regulates the protein levels of a subset of its targets, which are strongly enriched for fate-instructive chromatin regulators, and among them, histone chaperone ANP32B is experimentally verified and functionally relevant. Furthermore, NAT10-ac4C-ANP32B axis can modulate the chromatin landscape of their downstream genes (e.g., key regulators of Wnt and TGFβ pathways). Collectively, we show that NAT10 is an essential regulator of cellular plasticity, and its catalyzed mRNA cytidine acetylation represents a critical layer of epitranscriptomic modulation and uncover a previously unrecognized, direct cross-talk between epitranscriptomic modification and chromatin signaling during cell fate transitions.

Galectin-9 in cancer therapy: from immune checkpoint ligand to promising therapeutic target.

Galectin-9 (Gal-9) is a vital member of the galectin family, functioning as a multi-subtype galactose lectin with diverse biological roles. Recent research has revealed that Gal-9's interaction with tumors is an independent factor that influences tumor progression. Furthermore, Gal-9 in the immune microenvironment cross-talks with tumor-associated immune cells, informing the clarification of Gal-9's identity as an immune checkpoint. A thorough investigation into Gal-9's role in various cancer types and its interaction with the immune microenvironment could yield novel strategies for subsequent targeted immunotherapy. This review focuses on the latest advances in understanding the direct and indirect cross-talk between Gal-9 and hematologic malignancies, in addition to solid tumors. In addition, we discuss the prospects of Gal-9 in tumor immunotherapy, including its cross-talk with the ligand TIM-3 and its potential in immune-combination therapy.

Abstract B003: Communication is key: Understanding the modulating effect of immune cell crosstalk on T cell therapy function

Abstract T cell therapies have shown great promise, especially for the treatment of hematological malignancies, but this success has not been translatable for most solid tumors. Therefore, efforts have been put towards improving their efficacy, including optimization of the subset composition of the T cell product and development of strategies to overcome immunosuppression by the tumor microenvironment. Here, we combined advanced immune-organoid co-cultures and live 3D imaging to understand how different T cell subsets and immune cells in the tumor microenvironment can affect T cell therapy efficacy. We found that CD4+ and CD8+ subsets of therapeutic T cells cooperate to boost anti-tumor function, while tumor-associated macrophages (TAMs) have an opposing effect and reduce tumor-targeting by engineered T cells. Through our dynamic imaging-readout (Dekkers, Alieva Nature Biotech. 2022), we observed that a high level of interactions are involved in these modulating events, indicating direct crosstalk between the different cell types. To further investigate the impact of these interactions, we developed a 3D interactome mapping tool which enables us to profile all occurring cell-cell interactions in our 3D imaging datasets and determine their functional outcome. With this tool we identified a specific interaction between CD4+ and CD8+ T cells, where a subset of CD4+ T cells acquired a potent helper function over time by first engaging with tumor organoids and subsequently inducing CD8+ T cell target localization via direct contact. Multi-omics integration with single cell transcriptomic data allowed us to study the underlying molecular pathways involved in this interaction, leading to the identification of a crucial binding partner involved in this cooperative tumor-targeting behavior. Using the same strategy, we are currently investigating how interactions between TAMs and therapeutic T cells modulate their tumor-targeting activity. Together, these data highlight how cellular interactions can play an important role in engineered T cell function and we are currently exploring strategies to target these axes of immune cell communication to maximize therapy efficacy. Citation Format: Amber K.L. Wezenaar, Celina Honhoff, Helena van Eck, Elena Jiménez Curiel, Annelisa M Cornel, Femke C.A. Ringnalda, Tineke Aarts-Riemens, Jürgen Kuball, Zsolt Sebestyen, Hans Clevers, Stefan Nierkens, Johanna F. Dekkers, Ellen J. Wehrens, Maria Alieva, Anne C. Rios. Communication is key: Understanding the modulating effect of immune cell crosstalk on T cell therapy function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B003.

RNA‐Binding Proteins Musashi and Tau Aggresome in Alzheimer’s disease

AbstractBackgroundTauopathies, such as Alzheimer’s disease (AD), are characterized by the accumulation of toxic tau oligomers in the brain and accumulation of proteinaceous inclusions stored in cellular structures called aggresomes. Recent studies suggest that AD also exhibits pathology related to RNA binding proteins (RBPs), including Musashi proteins. Our lab has previously discovered that Musashi 1 (MSI1) and Musashi 2 (MSI2) aggregate in human AD brains and we have also visualized co‐localization with tau oligomers, suggesting toxic direct crosstalk between tau and MSI proteins. With this discovery and the growing evidence indicating MSI proteins as contributors to AD pathology, through their loss of functions it is crucial to elucidate how the interactions between misfolded tau and MSI influence tau aggregation and perhaps formation of cellular aggresomes.MethodIn this study, we characterize which tau oligomeric species, including phosphorylated and ubiquitinated ones, interact with MSI within the human AD brain (Braak Stage V‐VI). We investigate the deposition and cellular location of MSI and tau aggregates in AD and age‐matched control human cortical brain tissues by biochemical, immunohistochemistry and co‐immunofluorescence assays. Using high‐resolution microscopy and imaging analysis we measured the differences in size and morphology of MSI/tau co‐aggregates.ResultInvestigation of these co‐aggregates showed specific accumulation in neurons as well as in glial cells (astrocytes and microglia). We performed biochemical assays using commercial and in‐house antibodies to qualitatively investigate MSI1, MSI2, and tau across in AD brains. Co‐IF was performed to investigate the spatial and quantitative distribution of MSI1, MSI2, and tau aggregates. MSI/tau aggregate morphological characterization was conducted to profile their size and shape in AD cortices.ConclusionFor the first time, we show that the presence and quantity of tau oligomeric species and MSI co‐aggregates vary across the AD brains, showing inter‐subjects’ variability in size and deposition density. We also identify that tau conformers with MSI1 and MSI2 form different co‐aggregates in size and morphology quantifying their colocalization in high‐resolution 3D imaging. Further investigations are warranted to determine the temporal and regional distribution of these tau species in association with MSI proteins as well as the mechanisms underlying their aggregation.

Chlorophyll biosynthesis under the control of arginine metabolism

In natural environments, photosynthetic organisms adjust their metabolism to cope with the fluctuating availability of combined nitrogen sources, a growth-limiting factor. For acclimation, the dynamic degradation/synthesisof tetrapyrrolic pigments, as well as of the amino acid arginine, is pivotal; however, there has been no evidence that these processes could be functionally coupled. Using co-immunopurification and spectral shift assays, we found that in the cyanobacterium Synechocystis sp. PCC 6803, the arginine metabolism-related ArgD and CphB enzymes form protein complexes with Gun4, an essential protein for chlorophyll biosynthesis. Gun4 binds ArgD with high affinity, and the Gun4-ArgD complex accumulates in cells supplementedwith ornithine, a key intermediate of the arginine pathway. Elevated ornithine levels restricted de novo synthesis of tetrapyrroles, which arrested the recovery from nitrogen deficiency. Our data reveal a direct crosstalk between tetrapyrrole biosynthesis and arginine metabolism that highlights the importance of balancing photosynthetic pigment synthesis with nitrogen homeostasis.

SAT025 Characterization Of The Melatonin Type 1 Receptor / Cannabinoid Type 1 Receptor (MT1/CB1) Heteromers

Abstract Disclosure: Y. Cui: None. H. Cherdoud: None. G. Marsicano: None. L. Bellocchio: None. R. Jockers: None. O. Lahuna: None. Membrane receptors belonging to the G proteins coupled receptors (GPCRs), the largest family of proteins in the human genome with more than 800 members. GPCR functions were initially thought to take place at the plasma membrane upon binding of single monomeric receptor unit by their cognate ligand. This scheme was complexified after identification of cross-talks between GPCRs upon heteromerization and identification of functional GPCRs within intracellular organelles as nucleus or endosomes. Among a few others receptors, functional melatonin receptor type 1 (MT1) and cannabinoid receptor type 1 (CB1) were found in neuronal mitochondria, suggesting a cross-talk between them. Here, we are identifying and characterizing MT1/CB1 heteromers as a new GPCR heteromer. Previously we showed that coexpression of the MT1 and CB1 receptors induced the constitutive formation of MT1/CB1 heteromers using coimmunoprecipitation experiments on transiently transfected HEK293 cells. Using simultaneous PLA (Proximity Ligation Assay) and immunofluorescence experiments, we described the existence of intracellular MT1/CB1 heteromers associated with the mitochondrial network. Since then, we have further characterized the properties of MT1/CB1 heteromers. Coimmunoprecipitation experiments performed on cerebellum from wild-type, MT1 or CB1 knock-out mice enabled us to identify MT1/CB1 heteromers in vivo. We established stable monoclonal HEK293 cell lines expressing both receptors. Transmission Electronic Microscopy (TEM) and PLA experiments showed the presence of MT1/CB1 complexes not only intracellularly but also at the cell surface. Using PLA experiments we identified a penetrating interfering peptide that specifically dissociate the MT1/CB1 complex and thus open the door to experiments targeting specifically the complex. We were able to follow the melatonin-dependent internalization and trafficking of the MT1/CB1 complex from the plasma membrane to the late endosomes using a combination of Nanoluciferase complementation experiments and bystander BRET (Bioluminescence Resonance Energy Transfer). Costimulation of HE293 cells expressing both receptors with melatonin and the selective CB1 agonist ACEA (Arachidonyl-2'-chloroethylamide), a derivative of the endocannabinoid AEA (Arachidonylethanolamide or anandamide) resulted in a potentiation of the melatonin-dependent cAMP inhibition. Use of the identified interfering peptide will enable us to identify actions on signaling pathways resulting from a direct cross-talk between MT1 or CB1 subunits. Presentation: Saturday, June 17, 2023

Update on Adipose Tissue and Cancer.

Adipose tissue is the largest endocrine organ and an accepted contributor to overall energy homeostasis. There is strong evidence linking increased adiposity to the development of 13 types of cancer. With increased adiposity comes metabolic dysfunction and insulin resistance, and increased systemic insulin and glucose support the growth of many cancers, including those of the colon and endometrium. There is also an important direct crosstalk between adipose tissue and various organs. For instance, the healthy development and function of the mammary gland, as well as the development, growth, and progression of breast cancer, are heavily impacted by the breast adipose tissue in which breast epithelial cells are embedded. Cells of the adipose tissue are responsive to external stimuli, including overfeeding, leading to remodeling and important changes in the secretion of factors known to drive the development and growth of cancers. Loss of factors like adiponectin and increased production of leptin, endotrophin, steroid hormones, and inflammatory mediators have been determined to be important mediators of the obesity-cancer link. Obesity is also associated with a structural remodeling of the adipose tissue, including increased localized fibrosis and disrupted angiogenesis that contribute to the development and progression of cancers. Furthermore, tumor cells feed off the adipose tissue, where increased lipolysis within adipocytes leads to the release of fatty acids and stromal cell aerobic glycolysis leading to the increased production of lactate. Both have been hypothesized to support the higher energetic demands of cancer cells. Here, we aim to provide an update on the state of the literature revolving around the role of the adipose tissue in cancer initiation and progression.

Effects of chronic irisin treatment on brain monoamine levels in the hypothalamic and subcortical nuclei of adult male and female rats: An HPLC-ECD study

Monoaminergic systems are known to be involved in the pathophysiology of neuropsychiatric disorders and vegetative functions due to their established influence on hypothalamic and subcortical areas. These systems can be modulated by lifestyle factors, especially exercise, which is known to produce several beneficial effects on reproduction, brain health, and mental disorders. The fact that exercise is sensed by the brain shows that muscle-stimulated secretion of myokines allows direct crosstalk between the muscles and the brain. One of such exercise-induced beneficial effects on the brain is exhibited by irisin—a recently discovered PGC-1α-dependent adipo-myokine mainly secreted from skeletal muscle during exercise. Thus, we hypothesized that irisin may affect central monoamine levels and thus play an important role in the muscle–brain endocrine loop. To test this assertion, for 10 weeks, vehicle (deionized water) or 100 ng/kg irisin was injected intraperitoneally once a day to 12 male and 12 female rats after which the levels of monoamines and their metabolites were determined by HPLC-ECD. In the hypothalamic nuclei, irisin significantly decreased dopamine (DA) metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) (p < 0.05), DOPAC/DA ratio (p < 0.01) and noradrenaline (NA, p < 0.05) levels in the anteroventral periventricular nucleus (AVPV), and DOPAC and NA levels in the medial preoptic area (mPOA) (p < 0.05), having a crucial role in reproduction and sexual motivation, respectively. On the other hand, irisin significantly increased DOPAC levels in the lateral hypothalamic area (LHA) (p < 0.05), which acts as a hunger center, while it significantly decreased the levels of DA, NA, and its metabolite 3,4-dihydroxyphenylglycol (DHPG) in the ventromedial hypothalamic nucleus (VMH) as a known satiety center (p < 0.05). In nucleus accumbens (NaC), irisin significantly reduced 5-hydroxyindoleacetic acid (5-HIAA) levels (p < 0.05), which are implicated in autism spectrum disorder (ASD) physiopathology. It also significantly increased DA levels in this area, thus exhibiting positive effects on depression and sexual dysfunction in men. On the other hand, it significantly decreased serotonin (5-HT) (p < 0.01) and its metabolite 5-HIAA levels in the medial amygdala (MeA) (p < 0.05), indicating that it may play a role in social behaviors. Moreover, it significantly attenuated NA levels in the same subcortical area (p < 0.01), which is directly involved in stress-induced activation of the central noradrenergic system. These findings demonstrate for the first time that irisin induces significant changes in monoamine levels in many hypothalamic nuclei involved in feeding behavior and vegetative functions, as well as in subcortical nuclei related to neuropsychiatric disorders.

The DELLA-ABI4-HY5 module integrates light and gibberellin signals to regulate hypocotyl elongation

Plant growth is coordinately controlled by various environmental and hormonal signals, of which light and gibberellin (GA) signals are two critical factors with opposite effects on hypocotyl elongation. Although interactions between the light and GA signaling pathways have been studied extensively, the detailed regulatory mechanism of their direct crosstalk in hypocotyl elongation remains to be fully clarified. Previously, we reported that ABA INSENSITIVE 4 (ABI4) controls hypocotyl elongation through its regulation of cell-elongation-related genes, but whether it is also involved in GA signaling to promote hypocotyl elongation is unknown. In this study, we show that promotion of hypocotyl elongation by GA is dependent on ABI4 activation. DELLAs interact directly with ABI4 and inhibit its DNA-binding activity. In turn, ABI4 combined with ELONGATED HYPOCOTYL 5 (HY5), a key positive factor in light signaling, feedback regulates the expression of the GA2ox GA catabolism genes and thus modulates GA levels. Taken together, our results suggest that the DELLA-ABI4-HY5 module may serve as a molecular link that integrates GA and light signals to control hypocotyl elongation.

Abstract P1-13-18: EGFR signaling contributes to acquired resistance to CDK4/6 inhibitors in ER+ breast cancer cells in vitro and in vivo

Abstract Breast cancer driven by different hormone receptors, including estrogen receptor (ER+), is responsible for approximately 70-80% of cases among women. While endocrine therapy (ET) of ER+ primary tumors with antiestrogens or aromatase inhibitors is an effective first-line therapy, its success is limited by intrinsic and acquired resistance. Response to ET can be limited due to overexpression of cyclin D1, which promotes the activation of cyclin-dependent kinases 4 and 6 (CDK4/6). Selective inhibition of CDK4/6 and ER signaling is now standard-of-care therapy for ER+ metastatic breast cancer. CDK4/6 inhibitors (CDK4/6i), including palbociclib (PD), ribociclib, and abemaciclib, used in combination with ET, have shown improvement in progression-free survival compared to ET alone in the metastatic setting. However, the inevitable development of acquired resistance significantly limits the efficacy of this targeted therapy. Rewired signaling driven by different oncogenes, including the epidermal growth factor receptor (EGFR), overcomes the targeted inhibition of CDK4/6, which, in turn, allows cell cycle progression contributing to acquired resistance. We evaluated the expression and activity of EGFR in the panel of different matching pairs of ER- and ER+ CDK4/6i-sensitive (pS) and CDK4/6i-resistant (pR) breast cancer cell lines in vitro. Increased EGFR expression in ER+ MCF7/pR cells correlated with elevated ER phosphorylation, suggesting a direct cross-talk between EGFR and ER signaling in PD-resistant cells. Stimulation with EGF promoted ER activation, whereas estrogen stimulation promoted EGFR activation in ER+ pS and pR cell lines, indicating a direct cross-regulation between these molecular targets. Treatment of MCF7/pR cells with CDK4/6i resulted in elevated EGFR mRNA expression, confirming the dependency of resistant cells on EGFR signaling. Moreover, exposure to the EGFR inhibitors dramatically inhibited the proliferation of MCF7/pR cells compared to the parental cells, suggesting that EGFR could be an essential bypass signaling regulator contributing to resistance to CDK4/6i. We found that combined treatment with EGFRi and CDK4/6i significantly inhibited Rb phosphorylation in pR cells improving the effect of CDK4/6i on cell cycle arrest. We evaluated EGFR expression in MCF7/pS and pR mouse xenograft models in vivo. We observed a significant elevation in EGFR expression and phosphorylation in response to PD treatment, confirming the importance of EGFR signaling in maintaining the acquired resistance to CDK4/6i in ER+ breast cancer cells. Taken together, our findings suggest that EGFR signaling plays an important role in acquired resistance to CDK4/6i in ER+ breast cancer cells in vitro and in vivo and targeting EGFR with small-molecule inhibitors (like erlotinib or gefitinib) or chimeric monoclonal antibodies (i.e. cetuximab) can be a promising approach to improve CDK4/6i efficacy in breast cancer patients. Citation Format: Nadiia Lypova, Lilibeth Lanceta, Susan Daugherty, Jason Chesney, Yoannis Imbert-Fernandez. EGFR signaling contributes to acquired resistance to CDK4/6 inhibitors in ER+ breast cancer cells in vitro and in vivo. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-18.

Structured tracking of alcohol reinforcement (STAR) for basic and translational alcohol research

There is inherent tension between methodologies developed to address basic research questions in model species and those intended for preclinical to clinical translation: basic investigations require flexibility of experimental design as hypotheses are rapidly tested and revised, whereas preclinical models emphasize standardized protocols and specific outcome measures. This dichotomy is particularly relevant in alcohol research, which spans a diverse range of basic sciences in addition to intensive efforts towards understanding the pathophysiology of alcohol use disorder (AUD). To advance these goals there is a great need for approaches that facilitate synergy across basic and translational areas of nonhuman alcohol research. In male and female mice, we establish a modular alcohol reinforcement paradigm: Structured Tracking of Alcohol Reinforcement (STAR). STAR provides a robust platform for quantitative assessment of AUD-relevant behavioral domains within a flexible framework that allows direct crosstalk between translational and mechanistically oriented studies. To achieve cross-study integration, despite disparate task parameters, a straightforward multivariate phenotyping analysis is used to classify subjects based on propensity for heightened alcohol consumption and insensitivity to punishment. Combining STAR with extant preclinical alcohol models, we delineate longitudinal phenotype dynamics and reveal putative neuro-biomarkers of heightened alcohol use vulnerability via neurochemical profiling of cortical and brainstem tissues. Together, STAR allows quantification of time-resolved biobehavioral processes essential for basic research questions simultaneous with longitudinal phenotyping of clinically relevant outcomes, thereby providing a framework to facilitate cohesion and translation in alcohol research.

Human epicardial adipose tissue expresses glucose-dependent insulinotropic polypeptide, glucagon, and glucagon-like peptide-1 receptors as potential targets of pleiotropic therapies.

Human epicardial adipose tissue (EAT) plays a crucial role in the development and progression of coronary artery disease, atrial fibrillation, and heart failure. Microscopically, EAT is composed of adipocytes, nerve tissues, inflammatory, stromovascular, and immune cells. Epicardial adipose tissue is a white adipose tissue, albeit it also has brown fat-like or beige fat-like features. No muscle fascia divides EAT and myocardium; this allows a direct interaction and crosstalk between the epicardial fat and the myocardium. Thus, it might be a therapeutic target for pharmaceutical compounds acting on G-protein-coupled receptors, such as those for glucose-dependent insulinotropic polypeptide (GIP), glucagon (GCG), and glucagon-like peptide-1 (GLP-1), whose selective stimulation with innovative drugs has demonstrated beneficial cardiovascular effects. The precise mechanism of these novel drugs and their tissue and cellular target(s) need to be better understood. We evaluate whether human EAT expresses GIP, GCG, and GLP-1 receptors and whether their presence is related to EAT transcriptome. We also investigated protein expression and cell-type localization specifically for GIP receptor (GIPR) and glucagon receptor (GCGR). Epicardial adipose tissue samples were collected from 33 patients affected by cardiovascular diseases undergoing open heart surgery (90.9% males, age 67.2 ± 10.5 years mean ± SD). Microarray and immunohistochemistry analyses were performed. Microarray analysis showed that GIPR and GCGR messenger ribonucleic acids (mRNAs) are expressed in EAT, beyond confirming the previously found GLP-1 [3776 ± 1377 arbitrary unit (A.U.), 17.77 ± 14.91 A.U., and 3.41 ± 2.27 A.U., respectively]. The immunohistochemical analysis consistently indicates that GIPR and GCGR are expressed in EAT, mainly in macrophages, isolated, and in crown-like structures. In contrast, only some mature adipocytes of different sizes showed cytoplasmic immunostaining, similar to endothelial cells and pericytes in the capillaries and pre-capillary vascular structures. Notably, EAT GIPR is statistically associated with the low expression of genes involved in free fatty acid (FFA) oxidation and transport and those promoting FFA biosynthesis and adipogenesis (P < 0.01). Epicardial adipose tissue GCGR, in turn, is related to genes involved in FFA transport, mitochondrial fatty acid oxidation, and white-to-brown adipocyte differentiation, in addition to genes involved in the reduction of fatty acid biosynthesis and adipogenesis (P < 0.01). Having reported the expression of the GLP-1 receptor previously, here, we showed that GIPR and GCGR similarly present at mRNA and protein levels in human EAT, particularly in macrophages and partially adipocytes, suggesting these G-protein-coupled receptors as pharmacological targets on the ongoing innovative drugs, which seem cardiometabolically healthy well beyond their effects on glucose and body weight.