Luciferase Reporter Construct Research Articles

MicroRNA-Mediated Suppression of Glial Cell Line-Derived Neurotrophic Factor Expression Is Modulated by a Schizophrenia-Associated Non-Coding Polymorphism.

Plasma levels of glial cell line-derived neurotrophic factor (GDNF), a pivotal regulator of differentiation and survival of dopaminergic neurons, are reportedly decreased in schizophrenia. To explore the involvement of GDNF in the pathogenesis of the disease, a case-control association analysis was performed between five non-coding single nucleotide polymorphisms (SNP) across the GDNF gene and schizophrenia. Of them, the 'G' allele of the rs11111 SNP located in the 3' untranslated region (3'-UTR) of the gene was found to associate with schizophrenia. In silico analysis revealed that the rs11111 'G' allele might create binding sites for three microRNA (miRNA) species. To explore the significance of this polymorphism, transient co-transfection assays were performed in human embryonic kidney 293T (HEK293T) cells with a luciferase reporter construct harboring either the 'A' or 'G' allele of the 3'-UTR of GDNF in combination with the hsa-miR-1185-1-3p pre-miRNA. It was demonstrated that in the presence of the rs11111 'G' (but not the 'A') allele, hsa-miR-1185-2-3p repressed luciferase activity in a dose-dependent manner. Deletion of the miRNA binding site or its substitution with the complementary sequence abrogated the modulatory effect. Our results imply that the rs11111 'G' allele occurring more frequently in patients with schizophrenia might downregulate GDNF expression in a miRNA-dependent fashion.

A Variety of Mouse PYHIN Proteins Restrict Murine and Human Retroviruses.

PYHIN proteins are only found in mammals and play key roles in the defense against bacterial and viral pathogens. The corresponding gene locus shows variable deletion and expansion ranging from 0 genes in bats, over 1 in cows, and 4 in humans to a maximum of 13 in mice. While initially thought to act as cytosolic immune sensors that recognize foreign DNA, increasing evidence suggests that PYHIN proteins also inhibit viral pathogens by more direct mechanisms. Here, we examined the ability of all 13 murine PYHIN proteins to inhibit HIV-1 and murine leukemia virus (MLV). We show that overexpression of p203, p204, p205, p208, p209, p210, p211, and p212 strongly inhibits production of infectious HIV-1; p202, p207, and p213 had no significant effects, while p206 and p214 showed intermediate phenotypes. The inhibitory effects on infectious HIV-1 production correlated significantly with the suppression of reporter gene expression by a proviral Moloney MLV-eGFP construct and HIV-1 and Friend MLV LTR luciferase reporter constructs. Altogether, our data show that the antiretroviral activity of PYHIN proteins is conserved between men and mice and further support the key role of nuclear PYHIN proteins in innate antiviral immunity.

A Luciferase Reporter Assay to Detect Cellular Hypoxia In Vitro.

Hypoxia is a hallmark of ischemic cardiovascular diseases and solid malignant tumors. Cellular hypoxia induces numerous physiological and pathological processes, including hematopoiesis, angiogenesis, metabolic changes, cell growth, and apoptosis. Hypoxia-inducible factor-1 (HIF-1) binds to hypoxia response elements (HREs) to selectively induce the expression of various genes in response to hypoxia. Therefore, HREs have been used to develop hypoxia-targeted gene therapy.More than 70 pairs of HREs and hypoxia-inducible genes have been identified. The hypoxia-induced gene expression levels vary among HRE sequences depending on the number of HRE copies and oxygen levels. Most known HREs have not yet been thoroughly studied. Recent studies have revealed that the HRE-mediated effects of hypoxia are cell line-dependent. Herein we describe an in vitro method to investigate gene activation levels and characteristics based on varying the copy number of HREs in response to cellular hypoxia. We explain how to clone HREs into luciferase reporter constructs in the sense, antisense, and dual directions to measure luciferase expression for functional analyses.

Growth differentiation factor-15 stimulates the synthesis of corticotropin-releasing factor in hypothalamic 4B cells

Growth differentiation factor-15 (GDF15) is a stress-activated cytokine that regulates cell growth and inflammatory and stress responses. We previously reported the role and regulation of GDF15 in pituitary corticotrophs. Dexamethasone increases Gdf15 gene expression levels and production. GDF15 suppresses adrenocorticotropic hormone synthesis in pituitary corticotrophs and subsequently mediates the negative feedback effect of glucocorticoids. Here, we analyzed corticotropin-releasing factor (Crf) promoter activity in hypothalamic 4B cells transfected with promoter-driven luciferase reporter constructs. The effects of time and GDF15 concentration on Crf mRNA levels were analyzed using quantitative real-time polymerase chain reaction. Glial cell-derived neurotrophic factor family receptor α-like (GFRAL) protein is expressed in 4B cells. GDF15 increased Crf promoter activity and Crf mRNA levels in 4B cells. The protein kinase A and C pathways also contributed to the GDF15-induced increase in Crf gene expression. GDF15 stimulates GFRAL, subsequently increasing the phosphorylation of AKT, an extracellular signal-related kinase, and the cAMP response element-binding protein. Therefore, GDF15-dependent pathways may be involved in regulating Crf expression under stressful conditions in hypothalamic cells.

OR11-05 High-throughput Screening Identifies TR4 Modulators For Potential Therapeutic Application

Abstract Disclosure: S. Khowal: None. D. Zhang: None. R. Damoiseaux: None. K. Javaherian: None. A.P. Heaney: None. Cushing Disease (CD), due to an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor, leads to excess cortisol and is a life-threatening “orphan disease”. Although surgical removal can be effective, CD frequently recurs. Thereafter, repeat surgery, radiation therapy and/or bilateral adrenalectomy are not always successful and cause significant morbidity. Medical therapy has significantly improved but there is a clear unmet need for efficacious and safe therapies that target the pituitary corticotroph tumor itself to offer biochemical and tumor control. We previously demonstrated that the orphan testicular receptor 4 (TR4) regulates pro-opiomelanocortin gene transcription and ACTH secretion. Herein, we used the Flexi Vector system to identify TR4 modulators by fusing the TR4 LBD (380-615aa) to a 3’ GAL4 DBD to generate a chimeric fusion plasmid, pBIND-TR4-LBD. The pBIND-TR4-LBD construct and an exogenous luciferase reporter construct pGL4.35 (luc2P/9XGAL4UAS), containing 9 copies of a GAL4 responsive upstream activating sequence (UAS), were then transiently transfected into human embryonic kidney (HEK293) cells. Following treatment with TR4 ligands or modulators, our TR4-LBD undergoes a conformational change and recruits additional cofactors and RNA polymerase II to the complex to drive GAL4-directed firefly luciferase expression. In a high throughput screen (HTS) of 27,705 chemicals, we identified several potential “hit” TR4 modulators that induced or inhibited TR4 LBD-directed GAL4UAS-directed luciferase expression by >3 -fold. Compounds from the HTS included ATP-sensitive K+ and calcium channel blockers, steroid hormones and modulators of cell proliferation and inflammation and several agonist and antagonist “hit” compounds induced TR4 >10 fold or inhibited TR4 by 90% respectively with minimal cell toxicity (<10%). In silico analysis of interactions between the TR4 LBD and these potential ligands indicated that the GLU-534 & ARG-565 may be crucial residues involved in TR4 ligand binding. Dose-response curves to calculate EC50 of these potential TR4 hit compounds and mode-of-action characterization are in progress. Our studies have identified several potent TR4 agonistic and antagonistic compounds and lay the framework for the discovery of safe and efficacious lead TR4 ligands that can be further advanced as potential drug therapies for CD and other diseases. Presentation: Friday, June 16, 2023

MiR-199a-5p Decreases Esophageal Cancer Cell Proliferation Partially through Repression of Jun-B.

MicroRNA (miR)-199a-5p has been shown to function as a tumor suppressor in some malignancies but its role in esophageal cancer is poorly understood. To further explore its role in esophageal cancer, we sought to investigate the interaction between miR-199a-5p and Jun-B, an important component of the AP1 transcription factor, which contains a potential binding site for miR-199a-5p in its mRNA. We found that levels of miR-199a-5p are reduced in both human esophageal cancer specimens and in multiple esophageal cancer cell lines compared to esophageal epithelial cells. Jun-B expression is correspondingly elevated in these tumor specimens and in several cell lines compared to esophageal epithelial cells. Jun-B mRNA expression and stability, as well as protein expression, are markedly decreased following miR-199a-5p overexpression. A direct interaction between miR-199a-5p and Jun-B mRNA was confirmed by a biotinylated RNA-pull down assay and luciferase reporter constructs. Either forced expression of miR-199a-5p or Jun-B silencing led to a significant decrease in cellular proliferation as well as in AP-1 promoter activity. Our results provide evidence that miR-199a-5p functions as a tumor suppressor in esophageal cancer cells by regulating cellular proliferation, partially through repression of Jun B.

Tumor-Myeloid Cell Crosstalk Pathways Associated with Abscopal Responses in Breast Cancer

To identify predictive biomarkers for combined radiotherapy (RT) and immune checkpoint inhibitor (ICI) therapy, which can induce systemic anti-tumor immune responses (i.e., "abscopal" responses) in breast cancer. Four Trp53-/- Balb/c breast cancer syngeneic allograft models with low tumor mutational burden and resistance to dual ICI (anti-PD1 and anti-CTLA4, BioXcell) were used. Two lines exhibited abscopal responses to RT (8Gyx3) plus ICI combination therapy (i.e., "abscopal models"), whereas the other two did not (i.e., "non-abscopal models"). We performed spatial transcriptomics analysis (GeoMx whole transcriptome assay) of pan-cytokeratin positive (panCK+) tumor and tumor-adjacent CD45+ immune cell segments in orthotopically implanted tumors 10 days after initiating treatment with IgG control, RT alone, ICI alone, or RT/ICI combination (N>5 per group). Genes selectively induced by RT/ICI in panCK+ and CD45+ segments in abscopal models and were not induced in non-abscopal models were identified using two-tailed t-tests and FDR correction for multiple testing (FDR<5%). In vitro analyses of RT-induced secreted inflammatory signaling were conducted by exposing supernatant from irradiated (8Gy) tumor cells to a RAW264.7 macrophage cell line expressing an interferon-inducible luciferase reporter construct, followed by luciferase signal quantification (Invivogen). Hierarchical clustering of immune-related genes in CD45+ immune segments from breast cancer models with abscopal responses to RT/ICI revealed global differences in tumor-adjacent immune profiles at baseline and after RT/ICI treatment. Abscopal responsive breast cancer models at baseline showed an enrichment of myeloid cell subtypes expressing complement protein C1q, Ccl8, and Csf1r in tumor-adjacent CD45+ immune cells, and CD8+ T cells expressing Ccl5. 10 days after treatment with RT/ICI, tumor-adjacent CD45+ immune cells in abscopal models were enriched in Cxcl10, Irf7, and FcgRIV, whereas these genes were not induced in non-abscopal models. CyTOF analyses confirmed the induction of inflamed professional antigen presenting cells (CD11c+CD80+IA/IE+) by RT/ICI in abscopal models. Within panCK+ tumor segments, RT/ICI in abscopal models selectively induced Isg15 and Zbp1, both of which are involved in secreted inflammatory signaling. Consistently, RT-induced secreted inflammatory signaling from tumor cells to macrophages evaluated in vitro was significantly greater in abscopal models compared to non-abscopal models. Breast cancer models with abscopal responses to RT/ICI show increased Isg15- and Zbp1-dependent secreted inflammatory crosstalk that activates Cxcl10 and Irf7 expression in tumor-adjacent myeloid cells, which warrants further investigation as a potential predictive biomarker for combined RT/ICI therapy in breast cancer.

TRIM22 promotes the proliferation of glioblastoma cells by activating MAPK signaling and accelerating the degradation of Raf-1

The tripartite motif (TRIM) 22 and mitogen-activated protein kinase (MAPK) signaling pathways play critical roles in the growth of glioblastoma (GBM). However, the molecular mechanism underlying the relationship between TRIM22 and MAPK signaling remains unclear. Here, we found that TRIM22 binds to exon 2 of the sphingosine kinase 2 (SPHK2) gene. An ERK1/2-driven luciferase reporter construct identified TRIM22 as a potential activator of MAPK signaling. Knockout and overexpression of TRIM22 regulate the inhibition and activation of MAPK signaling through the RING-finger domain. TRIM22 binds to Raf-1, a negative regulator of MAPK signaling, and accelerates its degradation by inducing K48-linked ubiquitination, which is related to the CC and SPRY domains of TRIM22 and the C1D domain of Raf-1. In vitro and in vivo, an SPHK2 inhibitor (K145), an ERK1/2 inhibitor (selumetinib), and the nonphosphorylated mutant Raf-1S338A inhibited GBM growth. In addition, deletion of the RING domain and the nuclear localization sequence of TRIM22 significantly inhibited TRIM22-induced proliferation of GBM cells in vivo and in vitro. In conclusion, our study showed that TRIM22 regulates SPHK2 transcription and activates MAPK signaling through posttranslational modification of two critical regulators of MAPK signaling in GBM cells.

The 5′UTR of HCoV-OC43 adopts a topologically constrained structure to intrinsically repress translation

The emergence of SARS-CoV-2, which is responsible for the COVID-19 pandemic, has highlighted the need for rapid characterization of viral mechanisms associated with cellular pathogenesis. Viral UTRs represent conserved genomic elements that contribute to such mechanisms. Structural details of most CoV UTRs are not available, however. Experimental approaches are needed to allow for the facile generation of high-quality viral RNA tertiary structural models, which can facilitate comparative mechanistic efforts. By integrating experimental and computational techniques, we herein report the efficient characterization of conserved RNA structures within the 5'UTR of the HCoV-OC43 genome, a lab-tractable model coronavirus. We provide evidence that the 5'UTR folds into a structure with well-defined stem-loops (SLs) as determined by chemical probing and direct detection of hydrogen bonds by NMR. We combine experimental base-pair restraints with global structural information from SAXS to generate a 3D model that reveals that SL1-4 adopts a topologically constrained structure wherein SLs 3 and 4 coaxially stack. Coaxial stacking is mediated by short linker nucleotides and allows SLs 1 to 2 to sample different cojoint orientations by pivoting about the SL3,4 helical axis. To evaluate the functional relevance of the SL3,4 coaxial helix, we engineered luciferase reporter constructs harboring the HCoV-OC43 5'UTR with mutations designed to abrogate coaxial stacking. Our results reveal that the SL3,4 helix intrinsically represses translation efficiency since the destabilizing mutations correlate with increased luciferase expression relative to wildtype without affecting reporter mRNA levels, thus highlighting how the 5'UTR structure contributes to the viral mechanism.

Inducible Reporter Lines for Tissue-specific Monitoring of Drosophila Circadian Clock Transcriptional Activity

Organisms track time of day through the function of cell-autonomous molecular clocks. In addition to a central clock located in the brain, molecular clocks are present in most peripheral tissues. Circadian clocks are coordinated within and across tissues, but the manner through which this coordination is achieved is not well understood. We reasoned that the ability to track in vivo molecular clock activity in specific tissues of the fruit fly, Drosophila melanogaster, would facilitate an investigation into the relationship between different clock-containing tissues. Previous efforts to monitor clock gene expression in single flies in vivo have used regulatory elements of several different clock genes to dictate expression of a luciferase reporter enzyme, the activity of which can be monitored using a luminometer. Although these reporter lines have been instrumental in our understanding of the circadian system, they generally lack cell specificity, making it difficult to compare molecular clock oscillations between different tissues. Here, we report the generation of several novel lines of flies that allow for inducible expression of a luciferase reporter construct for clock gene transcriptional activity. We find that these lines faithfully report circadian transcription, as they exhibit rhythmic luciferase activity that is dependent on a functional molecular clock. Furthermore, we take advantage of our reporter lines' tissue specificity to demonstrate that peripheral molecular clocks are able to retain rhythmicity for multiple days under constant environmental conditions.

Novel 3' Proximal Replication Elements in Umbravirus Genomes.

The 3' untranslated regions (UTRs) of positive-strand RNA plant viruses commonly contain elements that promote viral replication and translation. The ~700 nt 3'UTR of umbravirus pea enation mosaic virus 2 (PEMV2) contains three 3' cap-independent translation enhancers (3'CITEs), including one (PTE) found in members of several genera in the family Tombusviridae and another (the 3'TSS) found in numerous umbraviruses and several carmoviruses. In addition, three 3' terminal replication elements are found in nearly every umbravirus and carmovirus. For this report, we have identified a set of three hairpins and a putative pseudoknot, collectively termed "Trio", that are exclusively found in a subset of umbraviruses and are located just upstream of the 3'TSS. Modification of these elements had no impact on viral translation in wheat germ extracts or in translation of luciferase reporter constructs in vivo. In contrast, Trio hairpins were critical for viral RNA accumulation in Arabidopsis thaliana protoplasts and for replication of a non-autonomously replicating replicon using a trans-replication system in Nicotiana benthamiana leaves. Trio and other 3' terminal elements involved in viral replication are highly conserved in umbraviruses possessing different classes of upstream 3'CITEs, suggesting conservation of replication mechanisms among umbraviruses despite variation in mechanisms for translation enhancement.

Cytochrome P450 Transcriptional Regulation by Testis-Specific Y-Encoded-Like Protein: Identification of Novel Upstream Transcription Factors.

Cytochrome P450s (CYPs) display significant inter-individual variation in expression, much of which remains unexplained by known CYP single-nucleotide polymorphisms (SNPs). Testis-specific Y-encoded-like proteins (TSPYLs) are transcriptional regulators for several drug-metabolizing CYPs including CYP3A4 However, transcription factors (TFs) that might influence CYP expression through an effect on TSPYL expression are unknown. Therefore, we studied regulators of TSPYL expression in hepatic cell lines and their possible SNP-dependent variation. Specifically, we identified candidate TFs that might influence TSPYL expression using the ENCODE ChIPseq database. Subsequently, the expression of TSPYL1/2/4 as well as that of selected CYP targets for TSPYL regulation were assayed in hepatic cell lines before and after knockdown of TFs that might influence CYP expression through TSPYL-dependent mechanisms. Those results were confirmed by studies of TF binding to TSPYL1/2/4 gene promoter regions. In hepatic cell lines, knockdown of the REST and ZBTB7A TFs resulted in decreased TSPYL1 and TSPYL4 expression and increased CYP3A4 expression, changes reversed by TSPYL1/4 overexpression. Potential binding sites for REST and ZBTB7A on the promoters of TSPYL1 and TSPYL4 were confirmed by chromatin immunoprecipitation. Finally, common SNP variants in upstream binding sites on the TSPYL1/4 promoters were identified and luciferase reporter constructs confirmed SNP-dependent modulation of TSPYL1/4 gene transcription. In summary, we identified REST and ZBTB7A as regulators of the expression of TSPYL genes which themselves can contribute to regulation of CYP expression and-potentially-of drug metabolism. SNP-dependent modulation of TSPYL transcription may contribute to individual variation in both CYP expression and-downstream-drug response phenotypes. SIGNIFICANCE STATEMENT: Testis-specific Y-encoded-like proteins (TSPYLs) are transcriptional regulators of cytochrome P450 (CYP) gene expression. Here, we report that variation in TSPYL expression as a result of the effects of genetically regulated TSPYL transcription factors is an additional factor that could result in downstream variation in CYP expression and potentially, as a result, variation in drug biotransformation.

The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFκB.

We previously showed that the matricellular protein CCN5 reverses established cardiac fibrosis (CF) through inducing apoptosis in myofibroblasts (MyoFBs) but not in cardiomyocytes or fibroblasts (FBs). In this study, we set out to elucidate the molecular mechanisms underlying CCN5-mediated selective apoptosis of MyoFBs. We first observed that the apoptotic protein p53 and the anti-apoptotic protein NFκB are simultaneously induced in MyoFBs. When the expression level of p53 was suppressed using a siRNA, CCN5 did not induce apoptosis in MyoFBs. By contrast, when NFκB signaling was inhibited using IKK VII, an IκB inhibitor, MyoFBs underwent apoptosis even in the absence of CCN5. SMAD7 is one of the downstream targets of CCN5 and it was previously shown to potentiate apoptosis in epithelial cells through inhibition of NFκB. In accordance with these reports, when the expression of SMAD7 was suppressed using a siRNA, NFκB signaling was enhanced, and CCN5 did not induce apoptosis. Lastly, we used a luciferase reporter construct to show that CCN5 positively regulated SMAD7 expression at the transcriptional level. Collectively, our data suggest that a delicate balance between the two mutually antagonistic proteins p53 and NFκB is maintained for MyoFBs to survive, and CCN5 tips the balance in favor of the apoptotic protein p53. This study provides insight into the anti-fibrotic activity of CCN5 during the regression of CF.

Interleukin 6 mediated activation of the mineralocorticoid receptor in the aldosterone-sensitive distal nephron.

Hypertension is characterized by increased sodium (Na+) reabsorption along the aldosterone-sensitive distal nephron (ASDN) as well as chronic systemic inflammation. Interleukin-6 (IL-6) is thought to be a mediator of this inflammatory process. Interestingly, increased Na+ reabsorption within the ASDN does not always correlate with increases in aldosterone (Aldo), the primary hormone that modulates Na+ reabsorption via the mineralocorticoid receptor (MR). Thus, understanding how increased ASDN Na+ reabsorption may occur independent of Aldo stimulation is critical. Here, we show that IL-6 can activate the MR by activating Rac1 and stimulating the generation of reactive oxygen species (ROS) with a consequent increase in thiazide-sensitive Na+ uptake. Using an in vitro model of the distal convoluted tubule (DCT2), mDCT15 cells, we observed nuclear translocation of eGFP-tagged MR after IL-6 treatment. To confirm the activation of downstream transcription factors, mDCT15 cells were transfected with mineralocorticoid response element (MRE)-luciferase reporter constructs; then treated with vehicle, Aldo, or IL-6. Aldosterone or IL-6 treatment increased luciferase activity that was reversed with MR antagonist cotreatment, but IL-6 treatment was reversed by Rac1 inhibition or ROS reduction. In both mDCT15 and mpkCCD cells, IL-6 increased amiloride-sensitive transepithelial Na+ current. ROS and IL-6 increased 22Na+ uptake via the thiazide-sensitive sodium chloride cotransporter (NCC). These results are the first to demonstrate that IL-6 can activate the MR resulting in MRE activation and that IL-6 increases NCC-mediated Na+ reabsorption, providing evidence for an alternative mechanism for stimulating ASDN Na+ uptake during conditions where Aldo-mediated MR stimulation may not occur.

Growth differentiation factor-15 modulates adrenocorticotropic hormone synthesis in murine AtT-20 corticotroph cells

Growth differentiation factor-15 (GDF15) is a stress-responsive cytokine that plays important roles in regulation of inflammatory responses, cell growth, and cell differentiation. However, the nature of these roles remains unclear. Here, we aimed to examine the regulatory effects of dexamethasone on Gdf15 expression in murine AtT-20 corticotroph cells. Human Gdf15 promoter-driven luciferase reporter constructs were transfected into corticotroph cells to analyze their promoter activity. The effects of time and concentration of dexamethasone on Gdf15 and proopiomelanocortin (Pomc) mRNA levels were assessed using quantitative real-time polymerase chain reaction. Dexamethasone induced Gdf15 transcription and mRNA levels as well as GDF15 production in transfected cells, whereas reduced the Pomc mRNA levels. GDF15 modulated adrenocorticotropic hormone (ACTH) synthesis, and the dexamethasone-mediated reduction in Pomc mRNA levels were partially relieved upon Gdf15 knockdown. We concluded that GDF15 modulated ACTH production in pituitary corticotrophs in an autocrine manner by suppressing Pomc expression and subsequently mediating the negative feedback effect of glucocorticoids, thereby contributing to pituitary stress response and homeostasis.

Regulation of Tissue Factor by CD44 Supports Coagulant Activity in Breast Tumor Cells

Simple SummaryMetastasis and thromboembolic complications are the main cause of cancer-associated death. An overexpression of coagulation factors, and particularly Tissue factor, by tumor cells is a key event implicated in this observed hypercoagulability. Tissue Factor is indeed a cellular initiator of the coagulation cascade which has been associated with aggressive tumor phenotypes such as those characteristic of Epithelial-Mesenchymal Transitions (EMTs) and Cancer Stem Cells (CSCs). Understanding molecular mechanisms controlling Tissue Factor overexpression in those tumor phenotypes is thus an important aspect of cancer research. We show here that CD44 (a transmembrane marker of CSC and EMT phenotypes) contributes to regulate TF expression at a transcriptional level, thereby supporting procoagulant properties in tumor cells that facilitate their metastatic spread.Previous work identified Tissue Factor (TF), a key activator of the coagulation cascade, as a gene induced in cellular contexts of Epithelial-Mesenchymal Transitions (EMTs), providing EMT+ Circulating Tumor Cells (CTCs) with coagulant properties that facilitate their metastatic seeding. Deciphering further molecular aspects of TF regulation in tumor cells, we report here that CD44 and TF coexpress in EMT contexts, and that CD44 acts as a regulator of TF expression supporting procoagulant properties and metastatic seeding. A transcriptional regulatory mechanism bridging CD44 to TF expression was further evidenced. Comparing different TF –promoter luciferase reporter constructs, we indeed found that the shortest -111 pb TF promoter fragment harboring three Specificity Protein 1 (Sp1) binding sites is still responsive to CD44 silencing. The observation that (i) mutation within Sp1 binding sites decreased the basal activity of the -111 pb TF promoter construct, (ii) CD44 silencing decreased Sp1 protein and mRNA levels and (iii) Sp1 silencing diminished TF expression further points to Sp1 as a key mediator linking CD44 to TF regulation. All together, these data thus report a transcriptional regulatory mechanism of TF expression by CD44 supporting procoagulant activity and metastatic competence of CTCs.

Keeping time in the dark: Potato diel and circadian rhythmic gene expression reveals tissue-specific circadian clocks.

The circadian clock is an internal molecular oscillator and coordinates numerous physiological processes through regulation of molecular pathways. Tissue‐specific clocks connected by mobile signals have previously been found to run at different speeds in Arabidopsis thaliana tissues. However, tissue variation in circadian clocks in crop species is unknown. In this study, leaf and tuber global gene expression in cultivated potato under cycling and constant environmental conditions was profiled. In addition, we used a circadian‐regulated luciferase reporter construct to study tuber gene expression rhythms. Diel and circadian expression patterns were present among 17.9% and 5.6% of the expressed genes in the tuber. Over 500 genes displayed differential tissue specific diel phases. Intriguingly, few core circadian clock genes had circadian expression patterns, while all such genes were circadian rhythmic in cultivated tomato leaves. Furthermore, robust diel and circadian transcriptional rhythms were observed among detached tubers. Our results suggest alternative regulatory mechanisms and/or clock composition is present in potato, as well as the presence of tissue‐specific independent circadian clocks. We have provided the first evidence of a functional circadian clock in below‐ground storage organs, holding important implications for other storage root and tuberous crops.

ESR1 F404 mutations and acquired resistance to fulvestrant in the plasmaMATCH study.

1009 Background: The selective estrogen receptor modulator (SERD) fulvestrant is commonly used to treat patients with hormone receptor positive advanced breast cancer, although potential mechanisms of acquired resistance are poorly understood. plasmaMATCH cohort A (NCT03182634) investigated the activity of fulvestrant in patients with activating ESR1 mutations in circulating tumor DNA (ctDNA). Here we present analysis of baseline and end-of-treatment (EOT) ctDNA to identify potential resistance mutations to fulvestrant. Methods: Paired baseline and EOT plasma samples from patients enrolled into plasmaMATCH underwent ctDNA sequencing (Guardant360, Guardant Health) to identify acquired mutations. For F404 analysis, MCF-7 cells were transiently transfected with estrogen receptor expression constructs containing either wildtype ESR1 (WT), single ESR1 mutations (D538G, E380Q, F404L), or compound mutations (D538G_F404L, E380Q_F404L), alongside an estrogen response element (ERE)-luciferase reporter construct. Transfected cells were treated with or without fulvestrant and ERE-luciferase activity compared. Results: Of 84 patients enrolled in cohort A, 69 had paired baseline and EOT sequencing. Patients with baseline ESR1 Y537S had shorter progression free survival (PFS), and Y537C longer PFS, than those wild-type for each respective mutation (p = 0.03 and p = 0.04). Patients frequently acquired mutations at EOT (n = 35, 51%), including potentially targetable mutations in 25% (including 3 PTEN, 3 BRCA1/2, 2 PIK3CA, 2 HER2, 1 BRAF). Three (4%) patients acquired ESR1 p.F404 mutations (F404L, F404I, F404V), with 7 mutations in total. Of 26 patients with PFS of ≥16 weeks, 3 patients (12%) acquired ESR1 F404. In 800 patients screened for entry to plasmaMATCH, one harbored a F404 mutation (0.13%), with a prior history of fulvestrant. F404 mutations resided in cis with E380Q in 6/7 assessable mutations. In vitro structural modelling revealed that ESR1 p.F404 resides within the ESR1 ligand binding domain, and contributes to estrogen and fulvestrant binding through a pi-stacking bond between the aromatic ring of phenylalanine and estrogen/fulvestrant, with all F404 mutations disrupting this bond. Transient transfection demonstrated that single mutations D538G, E380Q, F404L and wild-type ESR1 were sensitive to fulvestrant (p &lt; 0.0001, p = 0.0006, p = 0.04 and p = 0.0001), whereas compound mutations D538G_F404L and E380Q_F404L were resistant. Further investigation of relative sensitivity of the F404 mutant ESR1 to other anti-estrogens will be presented. Conclusions: We have identified a novel resistance mechanism to fulvestrant, with F404 mutations acquired in patients with pre-existing activating ESR1 mutations. F404 confers fulvestrant resistance through the loss of a pi-stacking bond and likely reduced fulvestrant binding affinity, identifying a new potential target to overcome endocrine therapy resistance.

LL-37 transports immunoreactive cGAMP to activate STING signaling and enhance interferon-mediated host antiviral immunity.

Cyclic 2',3'-GMP-AMP (cGAMP) binds to and activates stimulator of interferon genes (STING), which then induces interferons to drive immune responses against tumors and pathogens. Exogenous cGAMP produced by infected and malignant cells and synthetic cGAMP used in immunotherapy must traverse the cell membrane to activate STING in target cells. However, as an anionic hydrophilic molecule, cGAMP is not inherently membrane permeable. Here, we show that LL-37, a human host defense peptide, can function as a transporter of cGAMP. LL-37 specifically binds cGAMP and efficiently delivers cGAMP into target cells. cGAMP transferred by LL-37 activates robust interferon responses and host antiviral immunity in a STING-dependent manner. Furthermore, we report that LL-37 inducers vitamin D3 and sodium butyrate promote host immunity by enhancing endogenous LL-37 expression and its mediated cGAMP immune response. Collectively, our data uncover an essential role of LL-37 in innate immune activation and suggest new strategies for immunotherapy.

A β‐catenin‐TCF‐sensitive Locus Control Region Mediates GUCY2C Ligand Loss in Colorectal Cancer

BackgroundColorectal cancer most commonly arises from mutations in the tumor suppressor APC, or its downstream degradation target β‐catenin, initiating an oncogenic program of β‐catenin/TCF‐dependent nuclear transcription. Mechanisms coupling these mutations to tumorigenesis continue to be refined. The tumor suppressor axis regulated by the intestinal receptor guanylyl cyclase C (GUCY2C) and its paracrine ligands, guanylin and uroguanylin, contributes to intestinal homeostasis by regulating the proliferation, migration, and differentiation programs that maintain intestinal epithelial architecture. Furthermore, GUCY2C agonists oppose transformation in multiple mouse models of intestinal tumorigenesis. This signaling axis is among the earliest pathways silenced in tumorigenesis, reflecting an evolutionarily conserved pattern of GUCY2C retention, but ligand loss, in tumors. These observations suggest a pathophysiological model in which transformation orphans the receptor, silencing its signaling and lifting a block on tumorigenesis. Here, we examined the hypothesis that GUCY2C ligand loss arises from transcriptional silencing by β‐catenin/TCF.MethodsWe mapped the β‐catenin/TCF‐transcriptional program by RNA‐seq in four human colon cancer cell models. We then performed a comparative analysis of orthogonal approaches, including luciferase reporters, ChIP‐seq, CRISPR Cas9 knockout, and CRISPR epigenome editing to identify gene enhancers mediating GUCY2C ligand loss.ResultsRNA‐seq revealed a core β‐catenin/TCF‐transcriptional program of 1,289 genes, of which guanylin and uroguanylin were the seventh and twelfth most sensitive transcripts, reflecting transcriptional silencing. ChIP‐seq analyses of colon tissue revealed an evolutionarily conserved genomic locus encompassing the GUCY2C ligand genes, containing multiple regions of DNase hypersensitivity and H3K27ac enrichment, hallmarks of genetic enhancer loci. These markers were observed in normal colon, but not cancer tissue, consistent with enhancer inactivation and transcriptional silencing during transformation. ChIP‐seq in a colon cancer cell line revealed RNA Polymerase II and H3K27ac recruitment to the regions identified in human tissue, reflecting poised transcriptional machinery. Incorporation of the putative enhancer DNA into luciferase reporter constructs revealed a 2,683 bp locus control region (LCR) responsible for coordinated β‐catenin/TCF‐sensitive control of both GUCY2C ligand promoters. Both CRISPR‐Cas9 deletion of the LCR and epigenetic inactivation with a dCas9.KRAB repressor construct abolished β‐catenin/TCF‐sensitivity of GUCY2C ligand expression. Finally, LCR activation with a dCas9.VP64 transcriptional activator construct reconstituted GUCY2C ligand expression, overcoming gene silencing by β‐catenin/TCF.ConclusionsThis study reveals DNA elements regulating co‐repression of GUCY2C ligand transcription by β‐catenin/TCF, reflecting a novel step in tumorigenesis, and offers unique strategies to re‐establish hormone expression to oppose transformation.