The mast cell-T lymphocyte axis impacts cancer: Friend or foe?

Crosstalk between mast cells (MCs) and T lymphocytes (TLs) releases specific signals that create an environment conducive to tumor development. Conversely, they can protect against cancer by targeting tumor cells for destruction. Although their role in immunity and cancer is complex, their potential in anticancer strategies is often underestimated. When peripheral MCs are activated, they can affect cancer development. Tumor-infiltrating TLs may malfunction and contribute to aggressive cancer and poor prognoses. One promising approach for cancer patients is TL-based immunotherapies. Recent reports suggest that MCs modulate TL activity in solid tumors and may be a potential therapeutic layer in multitargeting anticancer strategies. Pharmacologically modulating MC activity can enhance the anticancer cytotoxic TL response in tumors. By identifying tumor-specific targets, it has been possible to genetically alter patients' cells into fully humanized anticancer cellular therapies for autologous transplantation, including the engineering of TLs and MCs to target and kill cancer cells. Hence, recent scientific evidence provides a broader understanding of MC-TL activity in cancer.

Resveratrol and 3,3'-Diindolylmethane Differentially Regulate Aryl Hydrocarbon Receptor and Estrogen Receptor Alpha Activity through Multiple Transcriptomic Targets in MCF-7 Human Breast Cancer Cells.

Inhibitory crosstalk between estrogen receptor alpha (ERα) and aryl hydrocarbon receptor (AHR) regulates 17ß-estradiol (E2)-dependent breast cancer cell signaling. ERα and AHR are transcription factors activated by E2 and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), respectively. Dietary ligands resveratrol (RES) and 3,3'diindolylmethane (DIM) also activate ERα while only DIM activates AHR and RES represses it. DIM and RES are reported to have anti-cancer and anti-inflammatory properties. Studies with genome-wide targets and AHR- and ERα-regulated genes after DIM and RES are unknown. We used chromatin immunoprecipitation with high-throughput sequencing and transcriptomics to study ERα as well as AHR coregulation in MCF-7 human breast cancer cells treated with DIM, RES, E2, or TCDD alone or E2+TCDD for 1 and 6 h, respectively. ERα bound sites after being DIM enriched for the AHR motif but not after E2 or RES while AHR bound sites after being DIM and E2+TCDD enriched for the ERE motif but not after TCDD. More than 90% of the differentially expressed genes closest to an AHR binding site after DIM or E2+TCDD also had an ERα site, and 60% of the coregulated genes between DIM and E2+TCDD were common. Collectively, our data show that RES and DIM differentially regulate multiple transcriptomic targets via ERα and ERα/AHR coactivity, respectively, which need to be considered to properly interpret their cellular and biological responses. These novel data also suggest that, when both receptors are activated, ERα dominates with preferential recruitment of AHR to ERα target genes.

Targeting AHR Increases Pancreatic Cancer Cell Sensitivity to Gemcitabine through the ELAVL1-DCK Pathway.

The aryl hydrocarbon receptor (AHR) is a transcription factor that is commonly upregulated in pancreatic ductal adenocarcinoma (PDAC). AHR hinders the shuttling of human antigen R (ELAVL1) from the nucleus to the cytoplasm, where it stabilises its target messenger RNAs (mRNAs) and enhances protein expression. Among these target mRNAs are those induced by gemcitabine. Increased AHR expression leads to the sequestration of ELAVL1 in the nucleus, resulting in chemoresistance. This study aimed to investigate the interaction between AHR and ELAVL1 in the pathogenesis of PDAC in vitro. AHR and ELAVL1 genes were silenced by siRNA transfection. The RNA and protein were extracted for quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis. Direct binding between the ELAVL1 protein and AHR mRNA was examined through immunoprecipitation (IP) assay. Cell viability, clonogenicity, and migration assays were performed. Our study revealed that both AHR and ELAVL1 inter-regulate each other, while also having a role in cell proliferation, migration, and chemoresistance in PDAC cell lines. Notably, both proteins function through distinct mechanisms. The silencing of ELAVL1 disrupts the stability of its target mRNAs, resulting in the decreased expression of numerous cytoprotective proteins. In contrast, the silencing of AHR diminishes cell migration and proliferation and enhances cell sensitivity to gemcitabine through the AHR-ELAVL1-deoxycytidine kinase (DCK) molecular pathway. In conclusion, AHR and ELAVL1 interaction can form a negative feedback loop. By inhibiting AHR expression, PDAC cells become more susceptible to gemcitabine through the ELAVL1-DCK pathway.

Association between AHR Expression and Immune Dysregulation in Pancreatic Ductal Adenocarcinoma: Insights from Comprehensive Immune Profiling of Peripheral Blood Mononuclear Cells.

Pancreatic cancer, particularly pancreatic ductal adenocarcinoma (PDAC), has an immune suppressive environment that allows tumour cells to evade the immune system. The aryl-hydrocarbon receptor (AHR) is a transcription factor that can be activated by certain exo/endo ligands, including kynurenine (KYN) and other tryptophan metabolites. Once activated, AHR regulates the expression of various genes involved in immune responses and inflammation. Previous studies have shown that AHR activation in PDAC can have both pro-tumorigenic and anti-tumorigenic effects, depending on the context. It can promote tumour growth and immune evasion by suppressing anti-tumour immune responses or induce anti-tumour effects by enhancing immune cell function. In this study involving 30 PDAC patients and 30 healthy individuals, peripheral blood samples were analysed. PDAC patients were categorized into Low (12 patients) and High/Medium (18 patients) AHR groups based on gene expression in peripheral blood mononuclear cells (PBMCs). The Low AHR group showed distinct immune characteristics, including increased levels of immune-suppressive proteins such as PDL1, as well as alterations in lymphocyte and monocyte subtypes. Functional assays demonstrated changes in phagocytosis, nitric oxide production, and the expression of cytokines IL-1, IL-6, and IL-10. These findings indicate that AHR's expression level has a crucial role in immune dysregulation in PDAC and could be a potential target for early diagnostics and personalised therapeutics.

Persistent organic pollutants promote aggressiveness in prostate cancer.

Increasing evidence points towards a causal link between exposure to persistent organic pollutants (POPs) with increased incidence and aggressivity of various cancers. Among these POPs, dioxin and PCB-153 are widely found in our environment and represent a significant source of contamination. Dioxin exposure has already been linked to cancer such as non-Hodgkin's lymphoma, but remains to be more extensively investigated in other cancers. Potential implications of dioxin and PCB-153 in prostate cancer progression spurred us to challenge both ex vivo and in vivo models with low doses of these POPs. We found that dioxin or PCB-153 exposure increased hallmarks of growth and metastasis of prostate cancer cells ex vivo and in grafted NOD-SCID mice. Exposure induced histopathological carcinoma-like patterns in the Ptenpc-/- mice. We identified up-regulation of Acetyl-CoA Acetyltransferase-1 (ACAT1) involved in ketone bodies pathway as a potential target. Mechanistically, genetic inhibition confirmed that ACAT1 mediated dioxin effect on cell migration. Using public prostate cancer datasets, we confirmed the deregulation of ACAT1 and associated gene encoded ketone bodies pathway enzymes such as OXCT1, BDH1 and HMGCL in advanced prostate cancer. To further explore this link between dioxin and ACAT1 deregulation, we analyzed a unique prostate-tumour tissue collection from the USA veterans exposed to agent orange, known to be highly contaminated by dioxin because of industrial production. We found that ACAT1 histoscore is significantly increased in exposed patients. Our studies reveal the implication of dioxin and PCB-153 to induce a prometastatic programme in prostate tumours and identify ACAT1 deregulation as a key event in this process.

Loss of PARP7 Increases Type I Interferon Signaling in EO771 Breast Cancer Cells and Prevents Mammary Tumor Growth by Increasing Antitumor Immunity.

PARP7 is a member of the ADP-ribosyltransferase diphtheria toxin-like (ARTD) family and acts as a repressor of type I interferon (IFN) signaling. PARP7 inhibition causes tumor regression by enhancing antitumor immunity, which is dependent on the stimulator of interferon genes (STING) pathway, TANK-binding kinase 1 (TBK1) activity, and cytotoxic CD8+ T cells. To better understand PARP7's role in cancer, we generated and characterized PARP7 knockout (Parp7KO) EO771 mouse mammary cancer cells in vitro and in a preclinical syngeneic tumor model using catalytic mutant Parp7H532A mice. Loss of PARP7 expression or inhibition of its activity increased type I IFN signaling, as well as the levels of interferon-stimulated gene factor 3 (ISGF3) and specifically unphosphorylated-ISGF3 regulated target genes. This was partly because PARP7's modification of the RelA subunit of nuclear factor κ-B (NF-κB). PARP7 loss had no effect on tumor growth in immunodeficient mice. In contrast, injection of wildtype cells into Parp7H532A mice resulted in smaller tumors compared with cells injected into Parp7+/+ mice. Parp7H532A mice injected with Parp7KO cells failed to develop tumors and those that developed regressed. Our data highlight the importance of PARP7 in the immune cells and further support targeting PARP7 for anticancer therapy.

Characterization of Intestinal Mesenchymal Stromal Cells From Patients With Inflammatory Bowel Disease for Autologous Cell Therapy.

Therapy with mesenchymal stromal cells (MSCs) has shown promise in inflammatory bowel disease-leveraging their immunosuppressive and regenerative properties. However, the potential immunogenic complications of allogenic MSCs sourced from different tissues raise concern. Thus, we assessed the fitness and functionality of autologous intestinal MSCs as a potential platform for cellular therapy. Mucosal biopsy-derived MSCs from Crohn's disease (n = 11), ulcerative colitis (n = 12), and controls (n = 14) were analyzed by microscopy and flow cytometry for doubling-time, morphology, differentiation potential, and immunophenotype. Gene expression, cell-subtype composition, along with surface marker and secretome changes after IFN-γ priming were measured by bulk and single-cell RNA sequencing coupled with a 30-plex Luminex panel. MSCs expanded ex vivo demonstrate canonical MSC markers, similar growth kinetics, and tripotency regardless of the patient phenotype. Global transcription patterns were similar at baseline though inflammatory bowel disease (IBD) rectal MSCs showed changes in select immunomodulatory genes. IFN-γ priming resulted in upregulation of shared immunoregulatory genes (particularly in PD-1 signaling) and overrode the transcriptional differences observed at baseline. Furthermore, MSCs secrete key immunomodulatory molecules at baseline and in response to IFN-γ including CXCL10, CXCL9, and MCP-1. Overall, MSCs from IBD patients have normal transcriptional and immunomodulatory properties with therapeutic potential and can be sufficiently expanded.

Longitudinal DNA methylation profiling of the rectal mucosa identifies cell-specific signatures of disease status, severity and clinical outcomes in ulcerative colitis cell-specific DNA methylation signatures of UC.

BACKGROUND: In peripheral blood, DNA methylation (DNAm) patterns in inflammatory bowel disease patients reflect inflammatory status rather than disease status. Here, we examined DNAm in diseased rectal mucosa from ulcerative colitis (UC) patients, focusing on constituent cell types with the goal of identifying therapeutic targets for UC other than the immune system. We profiled DNAm of rectal mucosal biopsies of pediatric UC at diagnosis (n = 211) and non-IBD control (n = 85) patients and performed epigenome-wide association studies (EWAS) of specific cell types to understand DNAm changes in epithelial, immune and fibroblast cells across disease states, course, and clinical outcomes. We also examined longitudinal analysis on follow-up samples (n = 73), and comparisons were made among patients with clinical outcomes including those undergoing colectomy versus those who did not. Additionally, we included RNA-seq from the same subjects to assess the impact of CpG sites on the transcription of nearby genes during the disease course. RESULTS: At diagnosis, UC rectal mucosa exhibited a lower proportion of epithelial cells and fibroblasts, and higher proportion of immune cells, in conjunction with variation in the DNAm pattern. While treatment had significant effects on the methylation signature of immune cells, its effects on fibroblasts and epithelial cells were attenuated. Individuals who required colectomy exhibited cell composition and DNAm patterns at follow-up more similar to disease onset than patients who did not require colectomy. Combining these results with gene expression profiles, we identify CpG sites whose methylation patterns are most consistent with a contribution to poor disease outcomes and could thus be potential therapeutic targets. CONCLUSIONS: Cell-specific epigenetic changes in the rectal mucosa in UC are associated with disease severity and outcome. Current therapeutics may more effectively target the immune than the epithelial and fibroblast compartments.

Identifying metabolic shifts in Crohn's disease using' omics-driven contextualized computational metabolic network models.

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. A clear gap in our existing CD diagnostics and current disease management approaches is the lack of highly specific biomarkers that can be used to streamline or personalize disease management. Comprehensive profiling of metabolites holds promise; however, these high-dimensional profiles need to be reduced to have relevance in the context of CD. Machine learning approaches are optimally suited to bridge this gap in knowledge by contextualizing the metabolic alterations in CD using genome-scale metabolic network reconstructions. Our work presents a framework for studying altered metabolic reactions between patients with CD and controls using publicly available transcriptomic data and existing gene-driven metabolic network reconstructions. Additionally, we apply the same methods to patient-derived ileal enteroids to explore the utility of using this experimental in vitro platform for studying CD. Furthermore, we have piloted an untargeted metabolomics approach as a proof-of-concept validation strategy in human ileal mucosal tissue. These findings suggest that in silico metabolic modeling can potentially identify pathways of clinical relevance in CD, paving the way for the future discovery of novel diagnostic biomarkers and therapeutic targets.

Structurally distinct PARP7 inhibitors provide new insights into the function of PARP7 in regulating nucleic acid-sensing and IFN-ß signaling.

The mono-ADP-ribosyltransferase PARP7 has emerged as a key negative regulator of cytosolic NA-sensors of the innate immune system. We apply a rational design strategy for converting a pan-PARP inhibitor into a potent selective PARP7 inhibitor (KMR-206). Consistent with studies using the structurally distinct PARP7 inhibitor RBN-2397, co-treatment of mouse embryonic fibroblasts with KMR-206 and NA-sensor ligands synergistically induced the expression of the type I interferon, IFN-ß. In mouse colon carcinoma (CT-26) cells, KMR-206 alone induced IFN-ß. Both KMR-206 and RBN-2397 increased PARP7 protein levels in CT-26 cells, demonstrating that PARP7's catalytic activity regulates its own protein levels. Curiously, treatment with saturating doses of KMR-206 and RBN-2397 achieved different levels of PARP7 protein, which correlated with the magnitude of type I interferon gene expression. These latter results have important implications for the mechanism of action of PARP7 inhibitors and highlights the usefulness of having structurally distinct chemical probes for the same target.

Assessing Cellular and Transcriptional Diversity of Ileal Mucosa Among Treatment-Naïve and Treated Crohn's Disease.

BACKGROUND: Crohn's disease is a lifelong disease characterized by chronic inflammation of the gastrointestinal tract. Defining the cellular and transcriptional composition of the mucosa at different stages of disease progression is needed for personalized therapy in Crohn's. METHODS: Ileal biopsies were obtained from (1) control subjects (n = 6), (2) treatment-naïve patients (n = 7), and (3) established (n = 14) Crohn's patients along with remission (n = 3) and refractory (n = 11) treatment groups. The biopsies processed using 10x Genomics single cell 5' yielded 139 906 cells. Gene expression count matrices of all samples were analyzed by reciprocal principal component integration, followed by clustering analysis. Manual annotations of the clusters were performed using canonical gene markers. Cell type proportions, differential expression analysis, and gene ontology enrichment were carried out for each cell type. RESULTS: We identified 3 cellular compartments with 9 epithelial, 1 stromal, and 5 immune cell subtypes. We observed differences in the cellular composition between control, treatment-naïve, and established groups, with the significant changes in the epithelial subtypes of the treatment-naïve patients, including microfold, tuft, goblet, enterocyte,s and BEST4+ cells. Surprisingly, fewer changes in the composition of the immune compartment were observed; however, gene expression in the epithelial and immune compartment was different between Crohn's phenotypes, indicating changes in cellular activity. CONCLUSIONS: Our study identified cellular and transcriptional signatures associated with treatment-naïve Crohn's disease that collectively point to dysfunction of the intestinal barrier with an increase in inflammatory cellular activity. Our analysis also highlights the heterogeneity among patients within the same disease phenotype, shining a new light on personalized treatment responses and strategies.

Mast cell-T cell axis alters development of colitis-dependent and colitis-independent colorectal tumours: potential for therapeutically targeting via mast cell inhibition.

BACKGROUND: Colorectal cancer (CRC) has a high mortality rate and can develop in either colitis-dependent (colitis-associated (CA)-CRC) or colitis-independent (sporadic (s)CRC) manner. There has been a significant debate about whether mast cells (MCs) promote or inhibit the development of CRC. Herein we investigated MC activity throughout the multistepped development of CRC in both human patients and animal models. METHODS: We analyzed human patient matched samples of healthy colon vs CRC tissue alongside conducting a The Cancer Genome Atlas-based immunogenomic analysis and multiple experiments employing genetically engineered mouse (GEM) models. RESULTS: Analyzing human CRC samples revealed that MCs can be active or inactive in this disease. An activated MC population decreased the number of tumor-residing CD8 T cells. In mice, MC deficiency decreased the development of CA-CRC lesions, while it increased the density of tumor-based CD8 infiltration. Furthermore, co-culture experiments revealed that tumor-primed MCs promote apoptosis in CRC cells. In MC-deficient mice, we found that MCs inhibited the development of sCRC lesions. Further exploration of this with several GEM models confirmed that different immune responses alter and are altered by MC activity, which directly alters colon tumorigenesis. Since rescuing MC activity with bone marrow transplantation in MC-deficient mice or pharmacologically inhibiting MC effects impacts the development of sCRC lesions, we explored its therapeutic potential against CRC. MC activity promoted CRC cell engraftment by inhibiting CD8+ cell infiltration in tumors, pharmacologically blocking it inhibits the ability of allograft tumors to develop. This therapeutic strategy potentiated the cytotoxic activity of fluorouracil chemotherapy. CONCLUSION: Therefore, we suggest that MCs have a dual role throughout CRC development and are potential druggable targets against this disease.

Aminoflavone upregulates putative tumor suppressor miR-125b-2-3p to inhibit luminal A breast cancer stem cell-like properties.

Metastatic breast cancer is incurable and often due to breast cancer stem cell (CSC)-mediated self-renewal. We previously determined that the aryl hydrocarbon receptor (AhR) agonist aminoflavone (AF) inhibits the expression of the CSC biomarker α6-integrin (ITGA6) to disrupt the formation of luminal (hormone receptor-positive) mammospheres (3D breast cancer spheroids). In this study, we performed miRNA-sequencing analysis of luminal A MCF-7 mammospheres treated with AF to gain further insight into the mechanism of AF-mediated anti-cancer and anti-breast CSC activity. AF significantly induced the expression of >70 microRNAs (miRNAs) including miR125b-2-3p, a predicted stemness gene regulator. AF-mediated miR125b-2-3p induction was validated in MCF-7 mammospheres and cells. miR125b-2-3p levels were low in breast cancer tissues irrespective of subtype compared to normal breast tissues. While miR125b-2-3p levels were low in MCF-7 cells, they were much lower in AHR100 cells (MCF-7 cells made unresponsive to AhR agonists). The miR125b-2-3p mimic decreased, while the antagomiR125b-2-3p increased the expression of stemness genes ITGA6 and SOX2 in MCF-7 cells. In MCF-7 mammospheres, the miR125b-2-3p mimic decreased only ITGA6 expression although the antagomiR125b-2-3p increased ITGA6, SOX2 and MYC expression. AntagomiR125b-2-3p reversed AF-mediated suppression of ITGA6. The miR125b-2-3p mimic decreased proliferation, migration, and mammosphere formation while the antagomiR125b-2-3p increased proliferation and mammosphere formation in MCF-7 cells. The miR125b-2-3p mimic also inhibited proliferation, mammosphere formation, and migration in AHR100 cells. AF induced AhR- and miR125b2-3p-dependent anti-proliferation, anti-migration, and mammosphere disruption in MCF-7 cells. Our findings suggest that miR125b-2-3p is a tumor suppressor and AF upregulates miR125b-2-3p to disrupt mammospheres via mechanisms that rely at least partially on AhR in luminal A breast cancer cells.

Aryl Hydrocarbon Receptor (AhR) Limits the Inflammatory Responses in Human Lung Adenocarcinoma A549 Cells via Interference with NF-κB Signaling.

Apart from its role in the metabolism of carcinogens, the aryl hydrocarbon receptor (AhR) has been suggested to be involved in the control of inflammatory responses within the respiratory tract. However, the mechanisms responsible for this are only partially known. In this study, we used A549 cell line, as a human model of lung alveolar type II (ATII)-like cells, to study the functional role of the AhR in control of inflammatory responses. Using IL-1ß as an inflammation inducer, we found that the induction of cyclooxygenase-2 and secretion of prostaglandins, as well as expression and release of pro-inflammatory cytokines, were significantly higher in the AhR-deficient A549 cells. This was linked with an increased nuclear factor-κB (NF-κB) activity, and significantly enhanced phosphorylation of its regulators, IKKα/ß, and their target IκBα, in the AhR-deficient A549 cells. In line with this, when we mimicked the exposure to a complex mixture of airborne pollutants, using an organic extract of reference diesel exhaust particle mixture, an exacerbated inflammatory response was observed in the AhR-deficient cells, as compared with wild-type A549 cells. Together, the present results indicate that the AhR may act as a negative regulator of the inflammatory response in the A549 model, via a direct modulation of NF-κB signaling. Its role(s) in the control of inflammation within the lung alveoli exposed to airborne pollutants, especially those which simultaneously activate the AhR, thus deserve further attention.

Reduced Colonic Mucosal Injury in 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Poly ADP-Ribose Polymerase (TIPARP/PARP7)-Deficient Mice.

Poly-ADP-ribose polymerases (PARPs) are important regulators of the immune system, including TCDD-inducible poly-ADP-ribose polymerase (TIPARP), also known as poly-ADP-ribose polymerase 7 (PARP7). PARP7 negatively regulates aryl hydrocarbon receptor (AHR) and type I interferon (IFN-I) signaling, both of which have been implicated in intestinal homeostasis and immunity. Since the loss of PARP7 expression increases AHR and IFN-I signaling, we used a murine dextran sulfate sodium (DSS)-induced colitis model to investigate the effect of PARP7 loss on DSS-induced intestinal inflammation. DSS-exposed Parp7-/- mice had less body weight loss, lower disease index scores, and reduced expression of several inflammation genes, including interleukin IL-6, C-x-c motif chemokine ligand 1 (Cxcl1), and lipocalin-2, when compared with wild-type mice. However, no significant difference was observed between genotypes in the colonic expression of the AHR target gene cytochrome P450 1A1 (Cyp1a1). Moreover, no significant differences in microbial composition were observed between the genotypes. Our findings demonstrate that the absence of PARP7 protein results in an impaired immune response to colonic inflammation and suggests that PARP7 may participate in the recruitment of immune cells to the inflammation site, which may be due to its role in IFN-I signaling rather than AHR signaling.

ADP-ribosyltransferases, an update on function and nomenclature.

ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD+ onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.

Analysis of the effects of aryl hydrocarbon receptor expression on cancer cell invasion via three-dimensional microfluidic invasion assays.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that binds to xenobiotics and activates expression of response elements to metabolize these compounds. The AHR pathway has been associated with a long list of diseases including cancer; however, it is debated whether AHR is tumorigenic or tumour-inhibiting. In particular, there are contradictory reports in the literature regarding the effects of AHR expression level on metastatic breast cancer. Here we used a 3D invasion assay called cell invasion in digital microfluidic microgel systems (CIMMS) to study the effect of AHR expression on invasion. In this study, MDA-MB-231 cells with stable knockout of AHR (AHRko) showed enhanced invasive characteristics and reduced proliferation, and cells with transient overexpression of AHR showed reduced invasiveness. Overexpression of AHR with a mutation in the DNA binding domain showed no difference in invasiveness compared to control, which suggests that the changes in invasiveness are related to the expression of AHR. CIMMS also allowed for extraction of sub-populations of invaded cells for RNA sequencing experiments. A comparison of the transcriptomes of invaded subpopulations of wild-type and AHRko cells identified 1809 genes that were differentially expressed, with enriched pathways including cell cycle, proliferation, survival, immunoproteasome activation, and activation of matrix metalloproteases. In sum, the data reported here for MDA-MB-231 cells suggests some new interpretations of the discrepancy in the literature on the role of AHR in breast cancer. We propose that the unique combination of functional discrimination with transcriptome profiling provided by CIMMS will be valuable for a wide range of mechanistic invasion-biology studies in the future.

Ileal Derived Organoids From Crohn's Disease Patients Show Unique Transcriptomic and Secretomic Signatures.

BACKGROUND & AIMS: We used patient-derived organoids (PDOs) to study the epithelial-specific transcriptional and secretome signatures of the ileum during Crohn's disease (CD) with varying phenotypes to screen for disease profiles and potential druggable targets. METHODS: RNA sequencing was performed on isolated intestinal crypts and 3-week-old PDOs derived from ileal biopsies of CD patients (n = 8 B1, inflammatory; n = 8 B2, stricturing disease) and non-inflammatory bowel disease (IBD) controls (n = 13). Differentially expressed (DE) genes were identified by comparing CD vs control, B1 vs B2, and inflamed vs non-inflamed. DE genes were used for computational screening to find candidate small molecules that could potentially reverse B1and B2 gene signatures. The secretome of a second cohort (n = 6 non-IBD controls, n = 7 CD, 5 non-inflamed, 2 inflamed) was tested by Luminex using cultured organoid conditioned medium. RESULTS: We found 90% similarity in both the identity and abundance of protein coding genes between PDOs and intestinal crypts (15,554 transcripts of 19,900 genes). DE analysis identified 814 genes among disease group (CD vs non-IBD control), 470 genes different between the CD phenotypes, and 5 false discovery rate correction significant genes between inflamed and non-inflamed CD. The PDOs showed both similarity and diversity in the levels and types of soluble cytokines and growth factors they released. Perturbagen analysis revealed potential candidate compounds to reverse B2 disease phenotype to B1 in PDOs. CONCLUSIONS: PDOs are similar at the transcriptome level with the in vivo epithelium and retain disease-specific gene expression for which we have identified secretome products, druggable targets, and corresponding pharmacologic agents. Targeting the epithelium could reverse a stricturing phenotype and improve outcomes.

Profiling non-coding RNA levels with clinical classifiers in pediatric Crohn's disease.

BACKGROUND: Crohn's disease (CD) is a heritable chronic inflammatory disorder. Non-coding RNAs (ncRNAs) play an important role in epigenetic regulation by affecting gene expression, but can also directly affect protein function, thus having a substantial impact on biological processes. We investigated whether non-coding RNAs (ncRNA) at diagnosis are dysregulated during CD at different CD locations and future disease behaviors to determine if ncRNA signatures can serve as an index to outcomes. METHODS: Using subjects belonging to the RISK cohort, we analyzed ncRNA from the ileal biopsies of 345 CD and 71 non-IBD controls, and ncRNA from rectal biopsies of 329 CD and 61 non-IBD controls. Sequence alignment was done (STAR package) using Human Genome version 38 (hg38) as reference panel. The differential expression (DE) analysis was performed with EdgeR package and DE ncRNAs were identified with a threshold of fold change (FC) > 2 and FDR < 0.05 after multiple test corrections. RESULTS: In total, we identified 130 CD specific DE ncRNAs (89 in ileum and 41 in rectum) when compared to non-IBD controls. Similarly, 35 DE ncRNAs were identified between B1 and B2 in ileum, whereas no differences among CD disease behaviors were noticed in rectum. We also found inflammation specific ncRNAs between inflamed and non-inflamed groups in ileal biopsies. Overall, we observed that expression of mir1244-2, mir1244-3, mir1244-4, and RN7SL2 were increased during CD, regardless of disease behavior, location, or inflammatory status. Lastly, we tested ncRNA expression at baseline as potential tool to predict the disease status, disease behaviors and disease inflammation at 3-year follow up. CONCLUSIONS: We have identified ncRNAs that are specific to disease location, disease behavior, and disease inflammation in CD. Both ileal and rectal specific ncRNA are changing over the course of CD, specifically during the disease progression in the intestinal mucosa. Collectively, our findings show changes in ncRNA during CD and may have a clinical utility in early identification and characterization of disease progression.

2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD)-Inducible Poly-ADP-Ribose Polymerase (TIPARP/PARP7) Catalytic Mutant Mice (TiparpH532A) Exhibit Increased Sensitivity to TCDD-Induced Hepatotoxicity and Lethality.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-adenosine diphosphate (ADP)-ribose polymerase (TIPARP/PARP7), an aryl hydrocarbon receptor (AHR) target gene and mono-ADP-ribosyltransferase, acts as part of a negative feedback loop to repress AHR signaling. This process is prevented by a single H532A mutation in TIPARP that destroys its catalytic activity. We hypothesized that the loss of TIPARP catalytic activity would increase sensitivity to TCDD-induced toxicity in vivo. To test this, we created a catalytically deficient mouse line (TiparpH532A) by introducing a single H532A mutation in TIPARP. Treatment of mouse embryonic fibroblasts or hepatocytes isolated from TiparpH532A mice confirmed the increased TCDD-induced expression of the AHR target genes Cyp1a1, Cyp1b1, and Tiparp. TiparpH532A mice given a single injection of 10 µg/kg TCDD, a nonlethal dose in Tiparp+/+ mice, did not survive beyond day 10. All Tiparp+/+ mice survived the 30-day treatment. TCDD-treated TiparpH532A mice displayed increased expression of AHR target genes, increased steatohepatitis and hepatotoxicity. Hepatic RNA-sequencing revealed 7-fold more differentially expressed genes in TiparpH532A mice than in Tiparp+/+ mice (4542 vs 647 genes) 6 days after TCDD treatment. Differentially expressed genes included genes involved in xenobiotic metabolism, lipid homeostasis and inflammation. Taken together, these data further support TIPARP as a critical negative regulator of AHR activity and show that loss of its catalytic activity is sufficient to increase sensitivity to TCDD-induced steatohepatitis and lethality. Since TIPARP inhibition has recently emerged as a potential anticancer therapy, the impact on AHR signaling, TCDD and polycyclic aromatic hydrocarbon toxicity will need to be carefully considered under conditions of therapeutic TIPARP inhibition.