Autocrine Feedback Loop Research Articles

E2F1-induced autocrine IL-6 inflammatory loop mediates cancer-immune crosstalk that predicts T cell phenotype switching and therapeutic responsiveness.

Melanoma is a metastatic, drug-refractory cancer with the ability to evade immunosurveillance. Cancer immune evasion involves interaction between tumor intrinsic properties and the microenvironment. The transcription factor E2F1 is a key driver of tumor evolution and metastasis. To explore E2F1's role in immune regulation in presence of aggressive melanoma cells, we established a coculture system and utilized transcriptome and cytokine arrays combined with bioinformatics and structural modeling. We identified an E2F1-dependent gene regulatory network with IL6 as a central hub. E2F1-induced IL-6 secretion unleashes an autocrine inflammatory feedback loop driving invasiveness and epithelial-to-mesenchymal transition. IL-6-activated STAT3 physically interacts with E2F1 and cooperatively enhances IL-6 expression by binding to an E2F1-STAT3-responsive promoter element. The E2F1-STAT3/IL-6 axis strongly modulates the immune niche and generates a crosstalk with CD4+ cells resulting in transcriptional changes of immunoregulatory genes in melanoma and immune cells that is indicative of an inflammatory and immunosuppressive environment. Clinical data from TCGA demonstrated that elevated E2F1, STAT3, and IL-6 correlate with infiltration of Th2, while simultaneously blocking Th1 in primary and metastatic melanomas. Strikingly, E2F1 depletion reduces the secretion of typical type-2 cytokines thereby launching a Th2-to-Th1 phenotype shift towards an antitumor immune response. The impact of activated E2F1-STAT3/IL-6 axis on melanoma-immune cell communication and its prognostic/therapeutic value was validated by mathematical modeling. This study addresses important molecular aspects of the tumor-associated microenvironment in modulating immune responses, and will contribute significantly to the improvement of future cancer therapies.

Looking for the philosopher's stone: Emerging approaches to target the hallmarks of aging in the skin.

Senescence and epigenetic alterations are two important hallmarks of cellular aging. During aging, cells subjected to stress undergo many cycles of damage and repair before finally entering either apoptosis or senescence, a permanent state of cell cycle arrest. The first biomarkers of senescence to be identified were increased ß-galactosidase activity and induction of p16INK4a. Another feature of senescent cells is the senescence-associated secretory phenotype (SASP), a complex secretome containing more than 80 pro-inflammatory factors including metalloproteinases, growth factors, chemokines and cytokines. The secretome is regulated through a dynamic process involving a self-amplifying autocrine feedback loop and activation of the immune system. Senescent cells play positive and negative roles depending on the composition of their SASP and may participate in various processes including wound healing and tumour suppression, as well as cell regeneration, embryogenesis, tumorigenesis, inflammation and finally aging. The SASP is also a biomarker of age, biological aging and age-related diseases. Recent advances in anti-age research have shown that senescence can be now prevented or delayed by clearing the senescent cells or mitigating the effects of SASP factors, which can be achieved by a healthy lifestyle (exercise and diet), and senolytics and senomorphics, respectively. An alternative is tissue rejuvenation, which can be achieved by stimulating aged stem cells and reprogramming deprogrammed aged cells. These non-clinical findings will open up new avenues of clinical research into the development of treatments capable of preventing or treating age-related pathologies in humans.

IL-6 facilitates cross-talk between epithelial cells and tumor- associated macrophages in Helicobacter pylori-linked gastric carcinogenesis

PurposeHelicobacter pylori (H. pylori) is a significant risk factor for development of gastric cancer (GC), one of the deadliest malignancies in the world. However, the mechanism by which H. pylori induces gastric oncogenesis remains unclear. Here, we investigated the function of IL-6 in gastric oncogenesis and macrophage-epithelial cell interactions. MethodsWe analyzed publicly available datasets to investigate the expression of IL-6 and infiltration of M2 macrophages in GC tissues, and determine the inter-cellular communication in the context of IL-6. Human gastric epithelial and macrophage cell lines (GES-1 and THP-1-derived macrophages, respectively) were used in mono- and co-culture experiments to investigate autocrine-and paracrine induction of IL-6 expression in response to H. pylori or IL-6 stimulation. ResultsWe found that IL-6 is highly expressed in GC and modulates survival. M2 macrophage infiltration is predominant in GC and drives an IL-6 mediated communication with gastric epithelium cells. In vitro, IL-6 triggers its own expression in GES-1 and THP-1-derived macrophages cells. In addition, these cell lines are able to upregulate each other's IL-6 levels in an autocrine fashion, which is enhanced by H. pylori stimulation. ConclusionThis study indicates that IL-6 in the tumor microenvironment is essential for intercellular communication. We show that H. pylori enhances an IL-6-driven autocrine and paracrine positive feedback loop between macrophages and gastric epithelial cells, which may contribute to gastric carcinogenesis.

Abstract A025: Autotaxin-lysolipid signaling suppresses a CCL11- eosinophil axis to promote pancreatic cancer progression

Abstract Among all cancer types, pancreatic ductal adenocarcinoma (PDAC) has one of the worst outcomes. In recent studies, metabolic and microenvironmental reprogramming in PDAC has been shown to facilitate tumor progression by promoting immune escape mechanisms, however these mechanisms are still poorly understood. Recently, our lab established that autotaxin (ATX) - lysophosphatidic acid (LPA) signaling in PDAC enhances tumorigenicity, but the immunomodulatory function of this signal pathway remains largely unexplored. We now show that ablation of the LPA-producing enzyme autotaxin (ATX) in PDAC cells can reduce tumor burden in mice and reprogram the tumor immune microenvironment by increasing tumor eosinophil infiltration. The functional role of eosinophils in the context of PDAC is a poorly understood area. The presence of eosinophils in pancreatic cancer has been reported in a few case reports: for example, prior studies identified neutrophils and eosinophils together as a group, expressing high levels of CD66b. We confirmed the presence of eosinophils in PDAC in mice as well as patients, and we found that rare patient samples with the highest intratumoral eosinophil abundance had the longest survival rates. Genetic or pharmacologic ATX inhibition increased intratumoral eosinophils, which promote tumor cell apoptosis locally and suppress tumor progression. When eosinophils were present, primary tumor growth was modestly reduced, but metastatic spread was greatly reduced. Mechanistically, ATX suppresses eosinophil accumulation via an autocrine feedback loop wherein ATX-LPA signaling negatively regulates activity of AP-1 transcription factor c-Jun, in turn suppressing expression of potent eosinophil chemoattractant CCL11 (Eotaxin-1). This is the first study that highlights the anti-tumor potential of eosinophils in pancreatic cancer. Citation Format: Sohinee Bhattacharyya, Chet Oon, Holly Sandborg, Terry Morgan, Mara H. Sherman. Autotaxin-lysolipid signaling suppresses a CCL11- eosinophil axis to promote pancreatic cancer progression [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 A025.

Autotaxin-lysolipid signaling suppresses a CCL11-eosinophil axis to promote pancreatic cancer progression.

Lipids and their modifying enzymes regulate diverse features of the tumor microenvironment and cancer progression. The secreted enzyme autotaxin (ATX) hydrolyzes extracellular lysophosphatidylcholine to generate the multifunctional lipid mediator lysophosphatidic acid (LPA) and supports the growth of several tumor types, including pancreatic ductal adenocarcinoma (PDAC). Here we show that ATX suppresses the accumulation of eosinophils in the PDAC microenvironment. Genetic or pharmacologic ATX inhibition increased the number of intratumor eosinophils, which promote tumor cell apoptosis locally and suppress tumor progression. Mechanistically, ATX suppresses eosinophil accumulation via an autocrine feedback loop, wherein ATX-LPA signaling negatively regulates the activity of the AP-1 transcription factor c-Jun, in turn suppressing the expression of the potent eosinophil chemoattractant CCL11 (eotaxin-1). Eosinophils were identified in human PDAC specimens, and rare individuals with high intratumor eosinophil abundance had the longest overall survival. Together with recent findings, this study reveals the context-dependent, immune-modulatory potential of ATX-LPA signaling in cancer.

Dexamethasone impairs the expression of antimicrobial mediators in lipopolysaccharide-activated primary macrophages by inhibiting both expression and function of interferon β

Glucocorticoids potently inhibit expression of many inflammatory mediators, and have been widely used to treat both acute and chronic inflammatory diseases for more than seventy years. However, they can have several unwanted effects, amongst which immunosuppression is one of the most common. Here we used microarrays and proteomic approaches to characterise the effect of dexamethasone (a synthetic glucocorticoid) on the responses of primary mouse macrophages to a potent pro-inflammatory agonist, lipopolysaccharide (LPS). Gene ontology analysis revealed that dexamethasone strongly impaired the lipopolysaccharide-induced antimicrobial response, which is thought to be driven by an autocrine feedback loop involving the type I interferon IFNβ. Indeed, dexamethasone strongly and dose-dependently inhibited the expression of IFNβ by LPS-activated macrophages. Unbiased proteomic data also revealed an inhibitory effect of dexamethasone on the IFNβ-dependent program of gene expression, with strong down-regulation of several interferon-induced antimicrobial factors. Surprisingly, dexamethasone also inhibited the expression of several antimicrobial genes in response to direct stimulation of macrophages with IFNβ. We tested a number of hypotheses based on previous publications, but found that no single mechanism could account for more than a small fraction of the broad suppressive impact of dexamethasone on macrophage type I interferon signaling, underlining the complexity of this pathway. Preliminary experiments indicated that dexamethasone exerted similar inhibitory effects on primary human monocyte-derived or alveolar macrophages.

Autocrine positive feedback of tumor necrosis factor from activated microglia proposed to be of widespread relevance in chronic neurological disease.

Over a decade's experience of post-stroke rehabilitation by administering the specific anti-TNF biological, etanercept, by the novel perispinal route, is consistent with a wide range of chronically diminished neurological function having been caused by persistent excessive cerebral levels of TNF. We propose that this TNF persistence, and cerebral disease chronicity, largely arises from a positive autocrine feedback loop of this cytokine, allowing the persistence of microglial activation caused by the excess TNF that these cells produce. It appears that many of these observations have never been exploited to construct a broad understanding and treatment of certain chronic, yet reversible, neurological illnesses. We propose that this treatment allows these chronically activated microglia to revert to their normal quiescent state, rather than simply neutralizing the direct harmful effects of this cytokine after its release from microglia. Logically, this also applies to the chronic cerebral aspects of various other neurological conditions characterized by activated microglia. These include long COVID, Lyme disease, post-stroke syndromes, traumatic brain injury, chronic traumatic encephalopathy, post-chemotherapy, post-irradiation cerebral dysfunction, cerebral palsy, fetal alcohol syndrome, hepatic encephalopathy, the antinociceptive state of morphine tolerance, and neurogenic pain. In addition, certain psychiatric states, in isolation or as sequelae of infectious diseases such as Lyme disease and long COVID, are candidates for being understood through this approach and treated accordingly. Perispinal etanercept provides the prospect of being able to treat various chronic central nervous system illnesses, whether they are of infectious or non-infectious origin, through reversing excess TNF generation by microglia.

Chronic stress accelerates glioblastoma progression via DRD2/ERK/β-catenin axis and Dopamine/ERK/TH positive feedback loop

BackgroundAfter diagnosis, glioblastoma (GBM) patients undertake tremendous psychological problems such as anxiety and depression, which may contribute to GBM progression. However, systematic study about the relationship between depression and GBM progression is still lacking.MethodsChronic unpredictable mild stress and chronic restrain stress were used to mimic human depression in mice. Human GBM cells and intracranial GBM model were used to assess the effects of chronic stress on GBM growth. Targeted neurotransmitter sequencing, RNA-seq, immunoblotting and immunohistochemistry were used to detect the related molecular mechanism.ResultsChronic stress promoted GBM progression and up-regulated the level of dopamine (DA) and its receptor type 2 (DRD2) in tumor tissues. Down-regulation or inhibition of DRD2 abolished the promoting effect of chronic stress on GBM progression. Mechanistically, the elevated DA and DRD2 activated ERK1/2 and consequently inhibited GSK3β activity, leading to β-catenin activation. Meanwhile, the activated ERK1/2 up-regulated tyrosine hydroxylase (TH) level in GBM cells and then promoted DA secretion, forming an autocrine positive feedback loop. Remarkably, patients with high-depression exhibited high DRD2 and β-catenin levels, which showed poor prognosis. Additionally, DRD2 specific inhibitor pimozide combined with temozolomide synergistically inhibited GBM growth.ConclusionsOur study revealed that chronic stress accelerates GBM progression via DRD2/ERK/β-catenin axis and Dopamine/ERK/TH positive feedback loop. DRD2 together with β-catenin may serve as a potential predictive biomarker for worse prognosis as well as therapeutic target of GBM patients with depression.

HILPDA is a lipotoxic marker in adipocytes that mediates the autocrine negative feedback regulation of triglyceride hydrolysis by fatty acids and alleviates cellular lipotoxic stress

BackgroundLipolysis is a key metabolic pathway in adipocytes that renders stored triglycerides available for use by other cells and tissues. Non-esterified fatty acids (NEFAs) are known to exert feedback inhibition on adipocyte lipolysis, but the underlying mechanisms have only partly been elucidated. An essential enzyme in adipocyte lipolysis is ATGL. Here, we examined the role of the ATGL inhibitor HILPDA in the negative feedback regulation of adipocyte lipolysis by fatty acids. MethodsWe exposed wild-type, HILPDA-deficient and HILPDA-overexpressing adipocytes and mice to various treatments. HILPDA and ATGL protein levels were determined by Western blot. ER stress was assessed by measuring the expression of marker genes and proteins. Lipolysis was studied in vitro and in vivo by measuring NEFA and glycerol levels. ResultsWe show that HILPDA mediates a fatty acid-induced autocrine feedback loop in which elevated intra- or extracellular fatty acids levels upregulate HILPDA by activation of the ER stress response and the fatty acid receptor 4 (FFAR4). The increased HILPDA levels in turn downregulate ATGL protein levels to suppress intracellular lipolysis, thereby maintaining lipid homeostasis. The deficiency of HILPDA under conditions of excessive fatty acid load disrupts this chain of events, leading to elevated lipotoxic stress in adipocytes. ConclusionOur data indicate that HILPDA is a lipotoxic marker in adipocytes that mediates a negative feedback regulation of lipolysis by fatty acids via ATGL and alleviates cellular lipotoxic stress.

Abstract 3531: Discrete extracellular matrix-secreting tumor cell subpopulations remodel the Ewing sarcoma tumor microenvironment to promote invasion

Abstract Ewing sarcoma (EwS) cells exist along a neuro-mesenchymal transcriptional continuum that is largely determined by transcriptional activity of the EWS::FLI1 fusion oncoprotein. Although EWS::FLI1-low state cells are more metastatic in experimental models, the role of these cells in established EwS tumor ecosystems is unknown. Here we have leveraged multimodal single-cell sequencing, spatial transcriptomics, and immunohistochemical profiling to characterize EwS cell subpopulations in cell lines, PDX models, and patient tumors. We identify CD73 as a marker of mesenchymal-high state EwS cells and show that CD73+ tumor cells share properties of experimentally-induced EWS::FLI1-low cells including altered cytoskeletons, enhanced migration and invasion, and increased expression of EWS::FLI1-repressed genes. However, CD73+ cells retain proliferative capacity and do not down-regulate the fusion or the EWS::FLI1-activated signature. Instead, CD73+ EwS cells selectively express a gene signature that is enriched in extracellular matrix (ECM) genes. Examination of xenograft and EwS patient biopsies confirms the existence of subpopulations of spatially and transcriptionally distinct ECM-producing tumor cells and shows them to be more prevalent in infiltrating regions. Importantly, the local tumor microenvironment (TME) surrounding these cells is rich in tumor-derived TNC, SPARC, and biglycan, ECM proteins that have been widely implicated in cancer metastatic progression. Significantly, CD73+ EwS cells also upregulate expression of ECM-sensing genes, including ITGA5 and ITGB1, suggesting potential to activate autocrine feedback loops. In support of this, interrupting outside-in ECM:integrin signaling cascades, through pharmacologic inhibition of FAK or SRC pathways, strongly reduces the invasive properties of CD73+ tumor spheroids in 3D collagen. Together these studies confirm the presence and functional importance of transcriptionally distinct tumor cell subpopulations in established EwS tumor ecosystems. In particular, our data reveal that highly mesenchymal tumor cells both generate and respond to pro-metastatic ECM proteins to support invasion. Studies are ongoing to determine the role of tumor cell-derived ECM on EwS progression and treatment resistance. Citation Format: Emma D. Wrenn, April A. Apfelbaum, Shireen Ganapathi, Erin R. Rudzinski, Xuemei Deng, Nicolas Garcia, Katherine Braun, Trisha Lipson, Shruti Bhise, Sami B. Kanaan, Olivia Waltner, Erika Newman, Scott Furlan, Elizabeth R. Lawlor. Discrete extracellular matrix-secreting tumor cell subpopulations remodel the Ewing sarcoma tumor microenvironment to promote invasion. [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 3531.

Autocrine activation of MAPK signaling mediates intrinsic tolerance to androgen deprivation in LY6D prostate cancer cells

The emergence of castration-resistant prostate cancer remains an area of unmet clinical need. We recently identified a subpopulation of normal prostate progenitor cells, characterized by an intrinsic resistance to androgen deprivation and expression of LY6D. We here demonstrate that conditional deletion of PTEN in the murine prostate epithelium causes an expansion of transformed LY6D+ progenitor cells without impairing stem cell properties. Transcriptomic analyses of LY6D+ luminal cells identified an autocrine positive feedback loop, based on the secretion of amphiregulin (AREG)-mediated activation of mitogen-activated protein kinase (MAPK) signaling, increasing cellular fitness and organoid formation. Pharmacological interference with this pathway overcomes the castration-resistant properties of LY6D+ cells with a suppression of organoid formation and loss of LY6D+ cells invivo. Notably, LY6D+ tumor cells are enriched in high-grade and androgen-resistant prostate cancer, providing clinical evidence for their contribution to advanced disease. Our data indicate that early interference with MAPK inhibitors can prevent progression of castration-resistant prostate cancer.

Abstract 2851: Moving on: Embracing γδ T-cell biology to create truly next generation immunotherapy concepts

Abstract Chimeric antigen receptor (CAR) modification of αβ T cells has revolutionized the field of oncology, driving the focus of attention to the immune system to target and fight malignancies. Currently available αβ T-cell therapies come with many challenges, including alloreactivity, off-target toxicities, cytokine release syndrome, limited survival of infused cells and the necessity to gene edit cells to overcome graft vs. host disease. Whilst some of these limitations can be overcome with complex and costly gene-engineering approaches, utilizing innate immune cells which are inherently non-HMC restricted and able to orchestrate wider immune responses might provide an alternative strategy avoiding many of these obstacles. Many groups developed protocols to grow and modify natural killer cells and invariant natural killer T-cells whilst others, including ours, focused on the development of Vδ1 γδ T-cell-based immunotherapies. Currently, these cell types are evaluated in the clinic in both non-engineered and engineered versions. Most of these engineering principles have been directly translated from the αβ CAR-T field without taking into consideration the biology of innate immune cells: they utilize αβ T-cell specific co-stimulatory molecules and signaling cassettes as well as armoring strategies developed for αβ T-cells. Vδ1 T-cells are tissue resident lymphocytes, which for most of their lifetime remain in epithelia rich tissues. They consistently survey tissues and monitor malignant transformation using a variety of natural cytotoxicity receptors, NK like receptors and the γδ TCR. In contrast to αβ T-cells, Vδ1 T-cells do not follow the ‘two-signal theory’: they must integrate multiple signals from an array of receptors in order to discriminate between healthy and malignant cells. We thus propose that CAR strategies exclusively utilizing CD3ζ activation domains are not using the full potential of innate immune cells for cellular immunotherapy. Another distinguishing feature of Vδ1 T-cells is the absence of traditional autocrine cytokine feedback loops seen in αβ T-cells. Our results demonstrate that traditional cytokine armoring strategies utilizing forced secretion of soluble or membrane-tethered IL-15 are detrimental to the biology of Vδ1 T-cells. By dissecting the IL-15 receptor biology we are now able to create cells that not only survive and grow in the absence of exogenous IL-15 but become more sensitive to endogenous levels. Applying γδ T-cell biology and an adapted understanding of the IL-15 pathway, we have now created novel engineering strategies to tailor specificity, potency, and proliferation of Vδ1 T-cells even in the presence of CAR whilst maintaining the cells’ inherent ability to discriminate between healthy and malignant cells. Preclinical evaluation of these concepts is ongoing with the aim to develop next generation tailored Vδ1T-cell immunotherapies. Citation Format: Jyothi Kumaran, Rajeev Karattil, André Simoes, Rebecca Alade, Mihil Patel, Liz Wood, Gonzalo Mercado Vico, Amy Lane, Sara Tamagno, Sarah Edwards, Andrea Venuso, Sam Illingworth, Alice Brown, Michael Koslowski, Istvan Kovacs, Oliver Nussbaumer. Moving on: Embracing γδ T-cell biology to create truly next generation immunotherapy concepts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2851.

IL-17 Receptor Signaling through IL-17A or IL-17F Is Sufficient to Maintain Innate Response and Control of Helicobacter pylori Immunopathogenesis.

IL-17R signaling is required for control of extracellular pathogens and is also implicated in development of chronic inflammatory processes. The response to the human pathogen Helicobacter pylori results in Th1 and Th17 cell activation and a chronic inflammatory process that can lead to adverse outcomes, such as gastric cancer. Previously, we identified IL-17RA as a requirement for the recruitment of neutrophils and control of H. pylori colonization in the gastric mucosa. Unexpectedly, H. pylori-infected Il17ra -/- mice had significantly more chronic inflammation than H. pylori-infected wild-type mice. In this study, human epithelial cell lines and murine models were used to investigate differential roles for IL-17A, IL-17F, and IL-17A/F during H. pylori infection. Moreover, the hypothesis that IL-17RA signaling, specifically in lymphocytes, provides an autocrine feedback loop that downregulates Th17 cytokine production was investigated. The data indicate that epithelial cells exhibit a stronger response to IL-17A and IL-17A/F than IL-17F, and that IL-17A and IL-17A/F can synergize with TNF and IL-22 to induce antimicrobial genes of gastric epithelial cells. In vivo deficiencies of IL-17A or IL-17F alone did not significantly change the immunopathological response to H. pylori, but if both cytokines were absent, a hyperinflammatory lymphocytic response developed. Using a cre/flox targeting approach for IL-17RA combined with infection, our findings demonstrate that increased chronic inflammation in Il17ra -/- mice was not attributed to a T cell-intrinsic defect. These data imply that IL-17A and IL-17F may have overlapping roles in maintenance of the gastric mucosal response to infection.

NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl- concentration.

The impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) activity induces intracellular chloride (Cl- ) accumulation. The anion Cl- , acting as a second messenger, stimulates the secretion of interleukin-1β (IL-1β), which starts an autocrine positive feedback loop. Here, we show that NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) are indirectly modulated by the intracellular Cl- concentration, showing maximal expression and activity at 75mM Cl- , in the presence of the ionophores nigericin and tributyltin. The expression of PYD and CARD domain containing (PYCARD/ASC) remained constant from 0 to 125mM Cl- . The CASP1 inhibitor VX-765 and the NLRP3 inflammasome inhibitor MCC950 completely blocked the Cl- -stimulated IL-1β mRNA expression and partially the IL-1β secretion. DCF fluorescence (cellular reactive oxygen species, cROS) and MitoSOX fluorescence (mitochondrial ROS, mtROS) also showed maximal ROS levels at 75mM Cl- , a response strongly inhibited by the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase (NOX) inhibitor GKT137831. These inhibitors also affected CASP1 and NLRP3 mRNA and protein expression. More importantly, the serum/glucocorticoid regulated kinase 1 (SGK1) inhibitor GSK650394, or its shRNAs, completely abrogated the IL-1β mRNA response to Cl- and the IL-1β secretion, interrupting the autocrine IL-1β loop. The results suggest that Cl- effects are mediated by SGK1, in which under Cl- modulation stimulates the secretion of mature IL-1β, in turn, responsible for the upregulation of ROS, CASP1, NLRP3 and IL-1β itself, through autocrine signalling.

Inhibition of relaxin autocrine signaling confers therapeutic vulnerability in ovarian cancer.

Ovarian cancer (OC) is the most deadly gynecological malignancy, with unmet clinical need for new therapeutic approaches. The relaxin peptide is a pleiotropic hormone with reproductive functions in the ovary. Relaxin induces cell growth in several types of cancer, but the role of relaxin in OC is poorly understood. Here, using cell lines and xenograft models, we demonstrate that relaxin and its associated GPCR RXFP1 form an autocrine signaling loop essential for OC in vivo tumorigenesis, cell proliferation, and viability. We determined that relaxin signaling activates expression of prooncogenic pathways, including RHO, MAPK, Wnt, and Notch. We found that relaxin is detectable in patient-derived OC tumors, ascites, and serum. Further, inflammatory cytokines IL-6 and TNF-α activated transcription of relaxin via recruitment of STAT3 and NF-κB to the proximal promoter, initiating an autocrine feedback loop that potentiated expression. Inhibition of RXFP1 or relaxin increased cisplatin sensitivity of OC cell lines and abrogated in vivo tumor formation. Finally, we demonstrate that a relaxin-neutralizing antibody reduced OC cell viability and sensitized cells to cisplatin. Collectively, these data identify the relaxin/RXFP1 autocrine loop as a therapeutic vulnerability in OC.

COX2 regulates senescence secretome composition and senescence surveillance through PGE2

SummarySenescent cells trigger their own immune-mediated destruction, termed senescence surveillance. This is dependent on the inflammatory senescence-associated secretory phenotype (SASP), which includes COX2, an enzyme with complex roles in cancer. The role COX2 plays during senescence surveillance is unknown. Here, we show that during RAS-induced senescence (RIS), COX2 is a critical regulator of SASP composition and senescence surveillance in vivo. COX2 regulates the expression of multiple inflammatory SASP components through an autocrine feedback loop involving its downstream product, prostaglandin E2 (PGE2), binding to EP4. During in vivo hepatocyte RIS, Cox2 is critical to tumor suppression, Cxcl1 expression, and immune-mediated senescence surveillance, partially through PGE2. Loss of Cox2 in RIS dysregulates the intrahepatic immune microenvironment, with enrichment of immunosuppressive immature myeloid cells and CD4+ regulatory T lymphocytes. Therefore, COX2 and PGE2 play a critical role in senescence, shaping SASP composition, promoting senescence surveillance and tumor suppression in the earliest stages of tumorigenesis.

Six1 Promotes Epithelial-Mesenchymal Transition in Bronchial Epithelial Cells via the TGFβ1/Smad Signalling Pathway

Introduction: The homeodomain transcription factor sine oculis homeobox homolog 1 (Six1) plays a crucial role in embryogenesis and is not expressed in normal adult tissue but is expressed in many pathological processes, including airway remodelling in asthma. The current study aimed to reveal the effects of Six1 in regulating the airway remodelling and its possible mechanism. Methods: A mouse model of ovalbumin-induced asthma-associated airway wall remodelling and a bronchial epithelial cell (16HBE) model of transforming growth factor β1 (TGFβ1)-induced epithelial-mesenchymal transition (EMT) were used to investigate the role of Six1. Then, 16HBE cells were transformed with Six1 expression vectors and treated with a TGFβ1 pathway inhibitor to determine the role of Six1 in EMT. The effect of Six1 and its possible mechanism were assessed by immunohistochemistry, RT-PCR, and Western blot. Results: Six1 expression was elevated in the lungs in an OVA mouse model of allergic asthma and in 16HBE cells treated with TGFβ1. Six1 overexpression promoted an EMT-like phenotype with a decreased protein expression of E-cadherin and increased protein expression of α-smooth muscle actin (α-SMA) as well as fibronectin in 16HBE cells; these effects appeared to promote TGFβ1 and phospho-Smad2 (pSmad2) production, which are the main products of the TGFβ1/Smad signalling pathway, which could be reduced by a TGFβ1 inhibitor. Conclusion: These data reveal that Six1 and TGFβ1 are potentially a part of an autocrine feedback loop that induces EMT, and these factors can be reduced by blocking the TGFβ1/Smad signalling pathway. As such, these factors may represent a promising novel therapeutic target for airway remodelling in asthma.

Autocrine Activation of MAPK-Signaling Mediates Intrinsic Tolerance to Androgen Deprivation in LY6D Prostate Cancer Cells

The emergence of castration resistant prostate cancer is associated with a high mortality and remains an area of unmet clinical need. We recently identified a rare subpopulation of normal prostate progenitor cells, characterized by an intrinsic resistance to androgen-deprivation and marked by the expression of LY6D. We here describe the underlying mechanisms driving castration-resistance of LY6D+ luminal progenitors and their contribution to advanced prostate cancer. We demonstrate that conditional deletion of PTEN in the murine prostate epithelium causes an expansion of transformed LY6D+ progenitor cells in proximal and distal regions of the prostate without impairing stem cell properties. Transcriptomic analyses of LY6D+ luminal cells identified an autocrine positive feed-back loop, based on the secretion of amphiregulin (AREG), further increasing cellular fitness and organoid formation. Pharmacological interference with AREG-activated MAPK-signaling overcomes the castration-resistant properties of LY6D+ cells with a near complete suppression of organoid formation. Notably, LY6D+ tumor cells are enriched in prostate specimens from high-grade and androgen-resistant prostate cancer, providing clinical evidence for their contribution to advanced and also metastatic disease. Our data indicate that the prospective identification of LY6D+ cells could allow for an early interference with MAPK-inhibitors to prevent the emergence of castration-resistant prostate cancer.

Unexpected Novel Findings That Caspase-1-KO Mice Are Poor Mobilizers and Engraft Poorly with Wild Type Bone Marrow Cells - Indicating a Presence of an Autocrine Feedback Loop Involving Interleukin 1b and Interleukin 18 Signaling That Potentiates Nlrp3 Inflammasome Activity, Both in HSPCs and in the BM Microenvironment for Optimal Stem Cell Trafficking

Background . Our recent results indicate that Nlrp3 inflammasome expressed in hematopoietic stem/progenitor cells (HSPCs) and in the bone marrow (BM) microenvironment are required for optimal mobilization of murine cells (Leukemia 2020 Jun;34(6):1512-1523, Stem Cell Rev Rep. 2019 Jun;15(3):391-403). Moreover, Nlrp3 inflammasome expressed in these cellular contexts are also required for normal homing and engraftment after hematopoietic transplantation (Stem Cell Rev and Rep (2020) in press. https://doi.org/10.1007/s12015-020-10005-w). Consistent with these results, Nlrp3-KO mice are poor mobilizers, cells from Nlrp3-KO mice engraft poorly, and, as transplant recipients, Nlrp3-KO mice show a delay in hematopoietic recovery after transplantation of normal BM cells. Activation of Nlrp3 inflammasomes leads to the release in caspase-1-dependent manner from HSPCs and cells in the BM microenvironment of two potent pro-inflammatory cytokines, interleukin 1b (IL-1b) and interleukin 18 (IL-18). Hypothesis. Based on the aforementioned results, we envisioned that the activated Nlrp3 inflammasome caspase-1 autocrine feedback loop involving IL-1bor IL-18 signaling potentiates HSPCs trafficking by amplifying Nlrp3 inflammasome activation in HSPCs and the BM microenvironment.Materials and Methods. We employed normal control and caspase-1-KO mice for mobilization and homing/engraftment experiments. Mice were mobilized with G-CSF or AMD3100 in the absence or presence of administered IL-1b or IL-18, and we measured i) the total number of white blood cells (WBCs) and ii) the number of clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. Next, caspase-1-KO cells were transplanted into lethally irradiated wild type animals, while caspase-1-KO mice were transplanted with wild type BMMNCs. We then evaluated homing and engraftment by measuring the number of PKH27-labeled cells, the number of clonogenic progenitors 24 hours after transplantation, and the numbers of day-12 CFU-S colonies and day-12 CFU-GM progenitors in the BM of recipient mice. In control experiments, to provide proof of our hypothesis, we also mobilized Nlrp3-KO mice with AMD3100 or G-CSF in the presence of administered IL-1b and IL-18. Results. We found that IL-1b or IL-18 alone mobilizes HSPCs and, significantly, that caspase-1-KO mice were poor mobilizers. Radiation chimera experiments revealed that this pro-mobilizing defect is dependent on the lack of caspase-1 in hematopoietic cells. To our surprise, administration of IL-1b and IL-18 did not improve G-CSF- or AMD3100-induced mobilization in Nlrp3-KO animals. We also found that both of these pro-inflammatory cytokines activate Nlrp3 inflammasomes in an Myd88-dependent manner. Moreover, HSPCs from caspase-1-KO mice show defective migration in response to a BM-released SDF-1 gradient and other supportive homing chemoattractants (sphingosine-1 phosphate; S1P and extracellular adenosine triphosphate; eATP). Finally, caspase-1-KO mice recipients were also engrafted poorly with normal HSPCs. To explain this phenomenon, we observed that the Nlrp3 inflammasome-caspase-1-IL-1b axis is important in upregulating SDF-1 and other key HSPC chemoattractants in the BM microenvironment conditioned for transplantation. Conclusions. We demonstrate for the first time that an Nlrp3 inflammasome-caspase-1-IL-1b/IL18 autocrine feedback mechanism operating in HSPCs and the BM microenvironment is involved in the normal trafficking of HSPCs during mobilization and homing/engraftment. This research demonstrates the unexpected role of caspase-1, which is situated downstream of the Nlrp3 inflammasome and directs the release of the potent pro-inflammatory cytokines IL-1b and IL18 that by feedback mechanism, in turn, activate Nlrp3 inflammasomes in HSPCs and the BM microenvironment, which is required for optimal trafficking of HSPCs. These results are important for understanding the novel casapase-1-mediated autocrine mechanisms involved not only in pharmacological mobilization but also in the inflammatory response of the organism to external challenges, such as infection and tissue/organ damage. Disclosures No relevant conflicts of interest to declare.

Loss of Mef2D function enhances TLR induced IL-10 production in macrophages

Mef2 transcription factors comprise a family of four different isoforms that regulate a number of processes including neuronal and muscle development. While roles for Mef2C and Mef2D have been described in B-cell development their role in immunity has not been extensively studied. In innate immune cells such as macrophages, TLRs drive the production of both pro- and anti-inflammatory cytokines. IL-10 is an important anti-inflammatory cytokine produced by macrophages and it establishes an autocrine feedback loop to inhibit pro-inflammatory cytokine production. We show here that macrophages from Mef2D knockout mice have elevated levels of IL-10 mRNA induction compared with wild-type cells following LPS stimulation. The secretion of IL-10 was also higher from Mef2D knockout macrophages and this correlated to a reduction in the secretion of TNF, IL-6 and IL-12p40. The use of an IL-10 neutralising antibody showed that this reduction in pro-inflammatory cytokine production in the Mef2D knockouts was IL-10 dependent. As the IL-10 promoter has previously been reported to contain a potential binding site for Mef2D, it is possible that the binding of other Mef2 isoforms in the absence of Mef2D may result in a higher activation of the IL-10 gene. Further studies with compound Mef2 isoforms would be required to address this. We also show that Mef2D is highly expressed in the thymus, but that loss of Mef2D does not affect thymic T-cell development or the production of IFNγ from CD8 T cells.