NR4A1 regulates expression of immediate early genes, suppressing replication stress in cancer.

Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3' UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.

The Lipogenic Regulator SREBP2 Induces Transferrin in Circulating Melanoma Cells and Suppresses Ferroptosis.

Circulating tumor cells (CTC) are shed by cancer into the bloodstream, where a viable subset overcomes oxidative stress to initiate metastasis. We show that single CTCs from patients with melanoma coordinately upregulate lipogenesis and iron homeostasis pathways. These are correlated with both intrinsic and acquired resistance to BRAF inhibitors across clonal cultures of BRAF-mutant CTCs. The lipogenesis regulator SREBP2 directly induces transcription of the iron carrier Transferrin (TF), reducing intracellular iron pools, reactive oxygen species, and lipid peroxidation, thereby conferring resistance to inducers of ferroptosis. Knockdown of endogenous TF impairs tumor formation by melanoma CTCs, and their tumorigenic defects are partially rescued by the lipophilic antioxidants ferrostatin-1 and vitamin E. In a prospective melanoma cohort, presence of CTCs with high lipogenic and iron metabolic RNA signatures is correlated with adverse clinical outcome, irrespective of treatment regimen. Thus, SREBP2-driven iron homeostatic pathways contribute to cancer progression, drug resistance, and metastasis. SIGNIFICANCE: Through single-cell analysis of primary and cultured melanoma CTCs, we have uncovered intrinsic cancer cell heterogeneity within lipogenic and iron homeostatic pathways that modulates resistance to BRAF inhibitors and to ferroptosis inducers. Activation of these pathways within CTCs is correlated with adverse clinical outcome, pointing to therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 521.

Stromal Microenvironment Shapes the Intratumoral Architecture of Pancreatic Cancer.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

Human anti-NKp46 antibody for studies of NKp46-dependent NK cell function and its applications for type 1 diabetes and cancer research.

Natural killer (NK) cells are innate lymphocytes that efficiently eliminate cancerous and infected cells. NKp46 is an important NK activating receptor shown to participate in recognition and activation of NK cells against pathogens, tumor cells, virally infected cells, and self-cells in autoimmune conditions, including type I and II diabetes. However, some of the NKp46 ligands are unknown and therefore investigating human NKp46 activity and its critical role in NK cell biology is problematic. We developed a unique anti-human NKp46 monocloncal antibody, denoted hNKp46.02 (02). The 02 mAb can induce receptor internalization and degradation. By binding to a unique epitope on a particular domain of NKp46, 02 lead NKp46 to lysosomal degradation. This downregulation therefore enables the investigation of all NKp46 activities. Indeed, using the 02 mAb we determined NK cell targets which are critically dependent on NKp46 activity, including certain tumor cells lines and human pancreatic beta cells. Most importantly, we showed that a toxin-conjugated 02 inhibits the growth of NKp46-positive cells; thus, exemplifying the potential of 02 in becoming an immunotherapeutic drug to treat NKp46-dependent diseases, such as, type I diabetes and NK and T cell related malignancies.

ß-Cell DNA Damage Response Promotes Islet Inflammation in Type 1 Diabetes.

Type 1 diabetes (T1D) is an autoimmune disease where pancreatic ß-cells are destroyed by islet-infiltrating T cells. Although a role for ß-cell defects has been suspected, ß-cell abnormalities are difficult to demonstrate. We show a ß-cell DNA damage response (DDR), presented by activation of the 53BP1 protein and accumulation of p53, in biopsy and autopsy material from patients with recently diagnosed T1D as well as a rat model of human T1D. The ß-cell DDR is more frequent in islets infiltrated by CD45+ immune cells, suggesting a link to islet inflammation. The ß-cell toxin streptozotocin (STZ) elicits DDR in islets, both in vivo and ex vivo, and causes elevation of the proinflammatory molecules IL-1ß and Cxcl10. ß-Cell-specific inactivation of the master DNA repair gene ataxia telangiectasia mutated (ATM) in STZ-treated mice decreases the expression of proinflammatory cytokines in islets and attenuates the development of hyperglycemia. Together, these data suggest that ß-cell DDR is an early event in T1D, possibly contributing to autoimmunity.

Sleeve Gastrectomy Improves Glycemia Independent of Weight Loss by Restoring Hepatic Insulin Sensitivity.

Bariatric surgery dramatically improves glycemic control, yet the underlying molecular mechanisms remain controversial because of confounding weight loss. We performed sleeve gastrectomy (SG) on obese and diabetic leptin receptor-deficient mice (db/db). One week postsurgery, mice weighed 5% less and displayed improved glycemia compared with sham-operated controls, and islets from SG mice displayed reduced expression of diabetes markers. One month postsurgery SG mice weighed more than preoperatively but remained near-euglycemic and displayed reduced hepatic lipid droplets. Pair feeding of SG and sham db/db mice showed that surgery rather than weight loss was responsible for reduced glycemia after SG. Although insulin secretion profiles from islets of sham and SG mice were indistinguishable, clamp studies revealed that SG causes a dramatic improvement in muscle and hepatic insulin sensitivity accompanied by hepatic regulation of hepatocyte nuclear factor-α and peroxisome proliferator-activated receptor-α targets. We conclude that long-term weight loss after SG requires leptin signaling. Nevertheless, SG elicits a remarkable improvement in glycemia through insulin sensitization independent of reduced feeding and weight loss.

Pancreatic ß-Cells Express the Fetal Islet Hormone Gastrin in Rodent and Human Diabetes.

ß-Cell failure in type 2 diabetes (T2D) was recently proposed to involve dedifferentiation of ß-cells and ectopic expression of other islet hormones, including somatostatin and glucagon. Here we show that gastrin, a stomach hormone typically expressed in the pancreas only during embryogenesis, is expressed in islets of diabetic rodents and humans with T2D. Although gastrin in mice is expressed in insulin+ cells, gastrin expression in humans with T2D occurs in both insulin+ and somatostatin+ cells. Genetic lineage tracing in mice indicates that gastrin expression is turned on in a subset of differentiated ß-cells after exposure to severe hyperglycemia. Gastrin expression in adult ß-cells does not involve the endocrine progenitor cell regulator neurogenin3 but requires membrane depolarization, calcium influx, and calcineurin signaling. In vivo and in vitro experiments show that gastrin expression is rapidly eliminated upon exposure of ß-cells to normal glucose levels. These results reveal the fetal hormone gastrin as a novel marker for reversible human ß-cell reprogramming in diabetes.

p16(Ink4a)-induced senescence of pancreatic beta cells enhances insulin secretion.

Cellular senescence is thought to contribute to age-associated deterioration of tissue physiology. The senescence effector p16(Ink4a) is expressed in pancreatic beta cells during aging and limits their proliferative potential; however, its effects on beta cell function are poorly characterized. We found that beta cell-specific activation of p16(Ink4a) in transgenic mice enhances glucose-stimulated insulin secretion (GSIS). In mice with diabetes, this leads to improved glucose homeostasis, providing an unexpected functional benefit. Expression of p16(Ink4a) in beta cells induces hallmarks of senescence--including cell enlargement, and greater glucose uptake and mitochondrial activity--which promote increased insulin secretion. GSIS increases during the normal aging of mice and is driven by elevated p16(Ink4a) activity. We found that islets from human adults contain p16(Ink4a)-expressing senescent beta cells and that senescence induced by p16(Ink4a) in a human beta cell line increases insulin secretion in a manner dependent, in part, on the activity of the mechanistic target of rapamycin (mTOR) and the peroxisome proliferator-activated receptor (PPAR)-γ proteins. Our findings reveal a novel role for p16(Ink4a) and cellular senescence in promoting insulin secretion by beta cells and in regulating normal functional tissue maturation with age.

PI3K/AKT/mTOR-dependent stabilization of oncogenic far-upstream element binding proteins in hepatocellular carcinoma cells.

UNLABELLED: Transcription factors of the far-upstream element-binding protein (FBP) family represent cellular pathway hubs, and their overexpression in liver cancer (hepatocellular carcinoma [HCC]) stimulates tumor cell proliferation and correlates with poor prognosis. Here we determine the mode of oncogenic FBP overexpression in HCC cells. Using perturbation approaches (kinase inhibitors, small interfering RNAs) and a novel system for rapalog-dependent activation of AKT isoforms, we demonstrate that activity of the phosphatidylinositol-4,5-biphosphate 3-kinase/AKT pathway is involved in the enrichment of nuclear FBP1 and FBP2 in liver cancer cells. In human HCC tissues, phospho-AKT significantly correlates with nuclear FBP1/2 accumulation and expression of the proliferation marker KI67. Mechanistic target of rapamycin (mTOR) inhibition or blockade of its downstream effector eukaryotic translation initiation factor 4E activity equally reduced FBP1/2 concentrations. The mTORC1 inhibitor rapamycin diminishes FBP enrichment in liver tumors after hydrodynamic gene delivery of AKT plasmids. In addition, the multikinase inhibitor sorafenib significantly reduces FBP levels in HCC cells and in multidrug resistance 2-deficient mice that develop HCC due to severe inflammation. Both FBP1/2 messenger RNAs are highly stable, with FBP2 being more stable than FBP1. Importantly, inhibition of phosphatidylinositol-4,5-biphosphate 3-kinase/AKT/mTOR signaling significantly diminishes FBP1/2 protein stability in a caspase-3/-7-dependent manner. CONCLUSION: These data provide insight into a transcription-independent mechanism of FBP protein enrichment in liver cancer; further studies will have to show whether this previously unknown interaction between phosphatidylinositol-4,5-biphosphate 3-kinase/AKT/mTOR pathway activity and caspase-mediated FBP stabilization allows the establishment of interventional strategies in FBP-positive HCCs.

Animal model studies indicate a candidate biomarker for sorafenib treatment of hepatocellular carcinoma.

In contrast to common genomic amplifications that support cancer cell growth by rewiring intracellular signaling, VEGFA amplification drives tumor cell proliferation via the tumor microenvironment. VEGFA amplification is present in a subset of mouse and human hepatocellular carcinomas (HCCs) that appear to be particularly sensitive to sorafenib treatment, indicating its potential value as a biomarker for HCC treatment.

Human and mouse VEGFA-amplified hepatocellular carcinomas are highly sensitive to sorafenib treatment.

UNLABELLED: Death rates from hepatocellular carcinoma (HCC) are steadily increasing, yet therapeutic options for advanced HCC are limited. We identify a subset of mouse and human HCCs harboring VEGFA genomic amplification, displaying distinct biologic characteristics. Unlike common tumor amplifications, this one seems to work via heterotypic paracrine interactions; stromal VEGF receptors (VEGFR), responding to tumor VEGF-A, produce hepatocyte growth factor (HGF) that reciprocally affects tumor cells. VEGF-A inhibition results in HGF downregulation and reduced proliferation, specifically in amplicon-positive mouse HCCs. Sorafenib-the first-line drug in advanced HCC-targets multiple kinases, including VEGFRs, but has only an overall mild beneficial effect. We found that VEGFA amplification specifies mouse and human HCCs that are distinctly sensitive to sorafenib. FISH analysis of a retrospective patient cohort showed markedly improved survival of sorafenib-treated patients with VEGFA-amplified HCCs, suggesting that VEGFA amplification is a potential biomarker for HCC response to VEGF-A-blocking drugs. SIGNIFICANCE: Using a mouse model of inflammation-driven cancer, we identified a subclass of HCC carrying VEGFA amplification, which is particularly sensitive to VEGF-A inhibition. We found that a similar amplification in human HCC identifies patients who favorably responded to sorafenib-the first-line treatment of advanced HCC-which has an overall moderate therapeutic efficacy.

Critical role for IL-1ß in DNA damage-induced mucositis.

ß-TrCP, the substrate recognition subunit of SCF-type ubiquitin ligases, is ubiquitously expressed from two distinct paralogs, targeting for degradation many regulatory proteins, among which is the NF-κB inhibitor IκB. To appreciate tissue-specific roles of ß-TrCP, we studied the consequences of inducible ablation of three or all four alleles of the E3 in the mouse gut. The ablation resulted in mucositis, a destructive gut mucosal inflammation, which is a common complication of different cancer therapies and represents a major obstacle to successful chemoradiation therapy. We identified epithelial-derived IL-1ß as the culprit of mucositis onset, inducing mucosal barrier breach. Surprisingly, epithelial IL-1ß is induced by DNA damage via an NF-κB-independent mechanism. Tissue damage caused by gut barrier disruption is exacerbated in the absence of NF-κB, with failure to express the endogenous IL-1ß receptor antagonist IL-1Ra upon four-allele loss. Antibody neutralization of IL-1ß prevents epithelial tight junction dysfunction and alleviates mucositis in ß-TrCP-deficient mice. IL-1ß antagonists should thus be considered for prevention and treatment of severe morbidity associated with mucositis.

Mutant KRAS is a druggable target for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) represents an unmet therapeutic challenge. PDA is addicted to the activity of the mutated KRAS oncogene which is considered so far an undruggable therapeutic target. We propose an approach to target KRAS effectively in patients using RNA interference. To meet this challenge, we have developed a local prolonged siRNA delivery system (Local Drug EluteR, LODER) shedding siRNA against the mutated KRAS (siG12D LODER). The siG12D LODER was assessed for its structural, release, and delivery properties in vitro and in vivo. The effect of the siG12D LODER on tumor growth was assessed in s.c. and orthotopic mouse models. KRAS silencing effect was further assessed on the KRAS downstream signaling pathway. The LODER-encapsulated siRNA was stable and active in vivo for 155 d. Treatment of PDA cells with siG12D LODER resulted in a significant decrease in KRAS levels, leading to inhibition of proliferation and epithelial-mesenchymal transition. In vivo, siG12D LODER impeded the growth of human pancreatic tumor cells and prolonged mouse survival. We report a reproducible and safe delivery platform based on a miniature biodegradable polymeric matrix, for the controlled and prolonged delivery of siRNA. This technology provides the following advantages: (i) siRNA is protected from degradation; (ii) the siRNA is slowly released locally within the tumor for prolonged periods; and (iii) the siG12D LODER elicits a therapeutic effect, thereby demonstrating that mutated KRAS is indeed a druggable target.

The expression of the beta cell-derived autoimmune ligand for the killer receptor nkp46 is attenuated in type 2 diabetes.

NK cells rapidly kill tumor cells, virus infected cells and even self cells. This is mediated via killer receptors, among which NKp46 (NCR1 in mice) is prominent. We have recently demonstrated that in type 1 diabetes (T1D) NK cells accumulate in the diseased pancreas and that they manifest a hyporesponsive phenotype. In addition, we found that NKp46 recognizes an unknown ligand expressed by beta cells derived from humans and mice and that blocking of NKp46 activity prevented diabetes development. Here we investigated the properties of the unknown NKp46 ligand. We show that the NKp46 ligand is mainly located in insulin granules and that it is constitutively secreted. Following glucose stimulation the NKp46 ligand translocates to the cell membrane and its secretion decreases. We further demonstrate by using several modalities that the unknown NKp46 ligand is not insulin. Finally, we studied the expression of the NKp46 ligand in type 2 diabetes (T2D) using 3 different in vivo models and 2 species; mice and gerbils. We demonstrate that the expression of the NKp46 ligand is decreased in all models of T2D studied, suggesting that NKp46 is not involved in T2D.

Tumor necrosis factor-α blocks differentiation and enhances suppressive activity of immature myeloid cells during chronic inflammation.

Elevated concentrations of tumor necrosis factor-α (TNF-α) are detected in pathologies characterized by chronic inflammation. Whether TNF-α plays a role in manipulating the host's immune system toward generating an immunosuppressive milieu, typical of ongoing chronic inflammation, is unclear. Here we showed that TNF-α exhibited a dual function during chronic inflammation: arresting differentiation of immature myeloid-derived suppressor cells (MDSCs) primarily via the S100A8 and S100A9 inflammatory proteins and their corresponding receptor (RAGE) and augmenting MDSC suppressive activity. These functions led to in vivo T and NK cell dysfunction accompanied by T cell antigen receptor ζ chain downregulation. Furthermore, administration of etanercept (TNF-α antagonist) during early chronic inflammatory stages reduced MDSCs' suppressive activity and enhanced their maturation into dendritic cells and macrophages, resulting in the restoration of in vivo immune functions and recovery of ζ chain expression. Thus, TNF has a fundamental role in promoting an immunosuppressive environment generated during chronic inflammation.

NKp46-mediated killing of human and mouse hepatic stellate cells attenuates liver fibrosis.

BACKGROUND: Liver fibrosis, which involves activation of hepatic stellate cells (HSC), is a major health problem and is the end outcome of all chronic liver diseases. The liver is populated with lymphocytes, among which are natural killer (NK) cells, whose activity is controlled by inhibitory and activating receptors. NKp46, one of the major NK activating receptors expressed by NK cells, is also a specific NK marker that discriminates NK cells from all other lymphocyte subsets. It recognises viral haemagglutinins and unknown cellular ligands. METHODS: The anti-fibrotic activity of the NKp46 receptor was assessed in vivo and in vitro using NKp46-deficient mice (NCR1(gfp/gfp)), the carbon tetrachloride model and in vitro NK killing assays. Primary murine and human HSC were stained for the expression of the NKp46 ligand using fusion proteins composed of the extracellular portions of the murine and human NKp46 receptors fused to human IgG1. RESULTS: It was shown that murine HSC express a ligand for the murine orthologue of the NKp46 receptor, NCR1. NCR1 inhibited liver fibrosis in vivo; in vitro, murine HSC were killed in an NCR1-dependent manner. In humans it was shown that human HSC also express a ligand for the human NKp46 receptor and that the killing of human HSC is NKp46 dependent. CONCLUSIONS: In addition to NKG2D, NKp46/NCR1 play an important role in inhibition of liver fibrosis. This suggests that fibrosis can be better controlled through the manipulation of NKp46 activity.

Epithelial microRNAs regulate gut mucosal immunity via epithelium-T cell crosstalk.

Colonic homeostasis entails epithelium-lymphocyte cooperation, yet many participants in this process are unknown. We show here that epithelial microRNAs mediate the mucosa-immune system crosstalk necessary for mounting protective T helper type 2 (T(H)2) responses. Abolishing the induction of microRNA by gut-specific deletion of Dicer1 (Dicer1(Δgut)), which encodes an enzyme involved in microRNA biogenesis, deprived goblet cells of RELMß, a key T(H)2 antiparasitic cytokine; this predisposed the host to parasite infection. Infection of Dicer1(Δgut) mice with helminths favored a futile T(H)1 response with hallmarks of inflammatory bowel disease. Interleukin 13 (IL-13) induced the microRNA miR-375, which regulates the expression of TSLP, a T(H)2-facilitating epithelial cytokine; this indicated a T(H)2-amplification loop. We found that miR-375 was required for RELMß expression in vivo; miR-375-deficient mice had significantly less intestinal RELMß, which possibly explains the greater susceptibility of Dicer1(Δgut) mice to parasites. Our findings indicate that epithelial microRNAs are key regulators of gut homeostasis and mucosal immunity.

Spermatogenesis rescue in a mouse deficient for the ubiquitin ligase SCF{beta}-TrCP by single substrate depletion.

beta-TrCP, the substrate recognition subunit of a Skp1-Cul1-F-box (SCF) ubiquitin ligase, is ubiquitously expressed from two distinct paralogs, targeting many regulatory proteins for proteasomal degradation. We generated inducible beta-TrCP hypomorphic mice and found that they are surprisingly healthy, yet have a severe testicular defect. We show that the two beta-TrCP paralogs have a nonredundant role in spermatogenesis. The testicular defect is tightly associated with cell adhesion failure within the seminiferous tubules and is fully reversible upon beta-TrCP restoration. Remarkably, testicular depletion of a single beta-TrCP substrate, Snail1, rescued the adhesion defect and restored spermatogenesis. Our studies highlight an unexpected functional reserve of this central E3, as well as a bottleneck in a specific tissue: a single substrate whose stabilization is incompatible with testicular differentiation.

S100A8 and S100A9 are novel nuclear factor kappa B target genes during malignant progression of murine and human liver carcinogenesis.

UNLABELLED: The nuclear factor-kappaB (NF-kappaB) signaling pathway has been recently shown to participate in inflammation-induced cancer progression. Here, we describe a detailed analysis of the NF-kappaB-dependent gene regulatory network in the well-established Mdr2 knockout mouse model of inflammation-associated liver carcinogenesis. Expression profiling of NF-kappaB-deficient and NF-kappaB-proficient hepatocellular carcinoma (HCC) revealed a comprehensive list of known and novel putative NF-kappaB target genes, including S100a8 and S100a9. We detected increased co-expression of S100A8 and S100A9 proteins in mouse HCC cells, in human HCC tissue, and in the HCC cell line Hep3B on ectopic RelA expression. Finally, we found a synergistic function for S100A8 and S100A9 in Hep3B cells resulting in a significant induction of reactive oxygen species (ROS), accompanied by enhanced cell survival. CONCLUSION: We identified S100A8 and S100A9 as novel NF-kappaB target genes in HCC cells during inflammation-associated liver carcinogenesis and provide experimental evidence that increased co-expression of both proteins supports malignant progression by activation of ROS-dependent signaling pathways and protection from cell death.

Human microRNAs regulate stress-induced immune responses mediated by the receptor NKG2D.

MICA and MICB are stress-induced ligands recognized by the activating receptor NKG2D. A microRNA encoded by human cytomegalovirus downregulates MICB expression by targeting a specific site in the MICB 3' untranslated region. As this site is conserved among different MICB alleles and a similar site exists in the MICA 3' untranslated region, we speculated that these sites are targeted by cellular microRNAs. Here we identified microRNAs that bound to these MICA and MICB 3' untranslated region sequences and obtained data suggesting that these microRNAs maintain expression of MICA and MICB protein under a certain threshold and facilitate acute upregulation of MICA and MICB during cellular stress. These microRNAs were overexpressed in various tumors and we demonstrate here that they aided tumor avoidance of immune recognition.