ABSTRACT
Tumour innervation is associated with worse patient outcomes in multiple cancers1,2, which suggests that it may regulate metastasis. Here we observed that highly metastatic mouse mammary tumours acquired more innervation than did less-metastatic tumours. This enhanced innervation was driven by expression of the axon-guidance molecule SLIT2 in tumour vasculature. Breast cancer cells induced spontaneous calcium activity in sensory neurons and elicited release of the neuropeptide substance P (SP). Using three-dimensional co-cultures and in vivo models, we found that neuronal SP promoted breast tumour growth, invasion and metastasis. Moreover, patient tumours with elevated SP exhibited enhanced lymph node metastatic spread. SP acted on tumoral tachykinin receptors (TACR1) to drive death of a small population of TACR1high cancer cells. Single-stranded RNAs (ssRNAs) released from dying cells acted on neighbouring tumoural Toll-like receptor 7 (TLR7) to non-canonically activate a prometastatic gene expression program. This SP- and ssRNA-induced Tlr7 gene expression signature was associated with reduced breast cancer survival outcomes. Therapeutic targeting of this neuro-cancer axis with the TACR1 antagonist aprepitant, an approved anti-nausea drug, suppressed breast cancer growth and metastasis in multiple models. Our findings reveal that tumour-induced hyperactivation of sensory neurons regulates multiple aspects of metastatic progression in breast cancer through a therapeutically targetable neuropeptide/extracellular ssRNA sensing axis.
ABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0176139.].
ABSTRACT
The gut microbiota affects tissue physiology, metabolism, and function of both the immune and nervous systems. We found that intrinsic enteric-associated neurons (iEANs) in mice are functionally adapted to the intestinal segment they occupy; ileal and colonic neurons are more responsive to microbial colonization than duodenal neurons. Specifically, a microbially responsive subset of viscerofugal CART+ neurons, enriched in the ileum and colon, modulated feeding and glucose metabolism. These CART+ neurons send axons to the prevertebral ganglia and are polysynaptically connected to the liver and pancreas. Microbiota depletion led to NLRP6- and caspase 11-dependent loss of CART+ neurons and impaired glucose regulation. Hence, iEAN subsets appear to be capable of regulating blood glucose levels independently from the central nervous system.
Subject(s)
Blood Glucose , Colon/innervation , Ganglia, Sympathetic/physiology , Gastrointestinal Microbiome/physiology , Ileum/innervation , Neurons/physiology , Animals , Anti-Bacterial Agents/pharmacology , Caspases, Initiator/genetics , Caspases, Initiator/physiology , Gastrointestinal Microbiome/drug effects , Liver/innervation , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/analysis , Neurons/chemistry , Pancreas/innervation , Receptors, Cell Surface/genetics , Receptors, Cell Surface/physiologyABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Connections between the gut and brain monitor the intestinal tissue and its microbial and dietary content1, regulating both physiological intestinal functions such as nutrient absorption and motility2,3, and brain-wired feeding behaviour2. It is therefore plausible that circuits exist to detect gut microorganisms and relay this information to areas of the central nervous system that, in turn, regulate gut physiology4. Here we characterize the influence of the microbiota on enteric-associated neurons by combining gnotobiotic mouse models with transcriptomics, circuit-tracing methods and functional manipulations. We find that the gut microbiome modulates gut-extrinsic sympathetic neurons: microbiota depletion leads to increased expression of the neuronal transcription factor cFos, and colonization of germ-free mice with bacteria that produce short-chain fatty acids suppresses cFos expression in the gut sympathetic ganglia. Chemogenetic manipulations, translational profiling and anterograde tracing identify a subset of distal intestine-projecting vagal neurons that are positioned to have an afferent role in microbiota-mediated modulation of gut sympathetic neurons. Retrograde polysynaptic neuronal tracing from the intestinal wall identifies brainstem sensory nuclei that are activated during microbial depletion, as well as efferent sympathetic premotor glutamatergic neurons that regulate gastrointestinal transit. These results reveal microbiota-dependent control of gut-extrinsic sympathetic activation through a gut-brain circuit.
Subject(s)
Gastrointestinal Microbiome/physiology , Intestines/innervation , Neurons/physiology , Sympathetic Nervous System/cytology , Sympathetic Nervous System/physiology , Animals , Dysbiosis/physiopathology , Female , Ganglia, Sympathetic/cytology , Ganglia, Sympathetic/physiology , Gastrointestinal Motility , Germ-Free Life , Intestines/microbiology , Male , Mice , Mice, Inbred C57BL , Models, Animal , Neural Pathways/physiology , Proto-Oncogene Proteins c-fos/metabolism , TranscriptomeABSTRACT
Enteric-associated neurons (EANs) are closely associated with immune cells and continuously monitor and modulate homeostatic intestinal functions, including motility and nutrient sensing. Bidirectional interactions between neuronal and immune cells are altered during disease processes such as neurodegeneration or irritable bowel syndrome. We investigated the effects of infection-induced inflammation on intrinsic EANs (iEANs) and the role of intestinal muscularis macrophages (MMs) in this context. Using murine models of enteric infections, we observed long-term gastrointestinal symptoms, including reduced motility and loss of excitatory iEANs, which was mediated by a Nlrp6- and Casp11-dependent mechanism, depended on infection history, and could be reversed by manipulation of the microbiota. MMs responded to luminal infection by upregulating a neuroprotective program via ß2-adrenergic receptor (ß2-AR) signaling and mediated neuronal protection through an arginase 1-polyamine axis. Our results identify a mechanism of neuronal death post-infection and point to a role for tissue-resident MMs in limiting neuronal damage.
Subject(s)
Intestinal Mucosa/immunology , Macrophages/immunology , Receptors, Adrenergic, beta-2/metabolism , Adrenergic Agents , Animals , Arginase/metabolism , Caspases, Initiator/immunology , Caspases, Initiator/metabolism , Enteric Nervous System/immunology , Enteric Nervous System/metabolism , Female , Gastrointestinal Diseases , Gastrointestinal Microbiome , Infections , Inflammation/immunology , Intestinal Mucosa/metabolism , Intestine, Small/immunology , Intestines/immunology , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Microbiota , Neurons/physiology , Receptors, Adrenergic, beta-2/immunology , Receptors, Cell Surface/immunology , Receptors, Cell Surface/metabolism , Signal TransductionABSTRACT
BACKGROUND: Despite improvements in cancer management, most pancreatic cancers are still diagnosed at an advanced stage. We have recently identified promoter DNA methylation of the genes ADAMTS1 and BNC1 as potential blood biomarkers of pancreas cancer. In this study, we validate this biomarker panel in peripheral cell-free tumor DNA of patients with pancreatic cancer. RESULTS: Sensitivity and specificity for each gene are as follows: ADAMTS1 87.2% and 95.8% (AUC = 0.91; 95% CI 0.71-0.86) and BNC1 64.1% and 93.7% (AUC = 0.79; 95% CI 0.63-0.78). When using methylation of either gene as a combination panel, sensitivity increases to 97.3% and specificity to 91.6% (AUC = 0.95; 95% CI 0.77-0.90). Adding pre-operative CA 19-9 values to the combined two-gene methylation panel did not improve sensitivity. Methylation of ADAMTS1 was found to be positive in 87.5% (7/8) of stage I, 77.8% (7/9) of stage IIA, and 90% (18/20) of stage IIB disease. Similarly, BNC1 was positive in 62.5% (5/8) of stage I patients, 55.6% (5/9) of stage IIA, and 65% (13/20) of patients with stage IIB disease. The two-gene panel (ADAMTS1 and/or BNC1) was positive in 100% (8/8) of stage I, 88.9% (8/9) of stage IIA, and 100% (20/20) of stage IIB disease. The sensitivity and specificity of the two-gene panel for localized pancreatic cancer (stages I and II), where the cancer is eligible for surgical resection with curative potential, was 94.8% and 91.6% respectively. Additionally, the two-gene panel exhibited an AUC of 0.95 (95% CI 0.90-0.98) compared to 57.1% for CA 19-9 alone. CONCLUSION: The methylation status of ADAMTS1 and BNC1 in cfDNA shows promise for detecting pancreatic cancer during the early stages when curative resection of the tumor is still possible. This minimally invasive blood-based biomarker panel could be used as a promising tool for diagnosis and screening in a select subset of high-risk populations.
Subject(s)
ADAMTS1 Protein/genetics , DNA Methylation , DNA-Binding Proteins/genetics , Pancreatic Neoplasms/diagnosis , Transcription Factors/genetics , Biomarkers, Tumor/blood , Biomarkers, Tumor/genetics , Cell-Free Nucleic Acids/genetics , Early Detection of Cancer , Epigenesis, Genetic , Female , Humans , Male , Neoplasm Staging , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Promoter Regions, Genetic , Sensitivity and SpecificityABSTRACT
PURPOSE: Chemotherapeutic resistance eventually develops in all patients with metastatic colorectal cancer (mCRC). Gene silencing through promoter demethylation is one potential reversible mechanism of resistance with administration of hypomethylating agents. We evaluated the safety and tolerability of guadecitabine and irinotecan in patients with mCRC previously treated with irinotecan. PATIENTS AND METHODS: In this 3+3 dose-escalation study, patients with mCRC previously exposed to irinotecan received guadecitabine days 1 to 5 of a 28-day cycle and irinotecan 125 mg/m2 days 8 and 15 [dose level (DL) 1, guadecitabine 45 mg/m2; DL -1: guadecitabine 30 mg/m2; DL -1G: guadecitabine 30 mg/m2 with growth factor support (GFS); DL 1G: guadecitabine 45 mg/m2 with GFS]. RESULTS: Twenty-two patients were treated across four DLs. Dose-limiting toxicities were neutropenic fever (DL 1 and -1G), biliary drain infection (DL -1), colonic obstruction (DL -1), and severe dehydration (DL 1G). Most common toxicities were neutropenia (82% any grade, 77% Grade 3/4), neutropenic fever (23%), leukopenia (73% any grade, 50% Grade 3/4), and injection site reactions (64% total, 0% Grade 3/4). Patients received a median of 4.5 cycles of treatment; 12/17 evaluable patients had stable disease as best response, with one having initial disease progression but subsequently durable partial response. Circulating tumor DNA showed decrease in global demethylation by LINE-1 after treatment. CONCLUSIONS: We report the first study of chemo-priming with epigenetic therapy in gastrointestinal cancers. Guadecitabine 45 mg/m2 and irinotecan 125 mg/m2 with GFS was safe and tolerable in patients with mCRC, with early indication of benefit. These data have provided the basis for an ongoing phase II randomized, multicenter trial.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Azacitidine/administration & dosage , Azacitidine/analogs & derivatives , Azacitidine/pharmacokinetics , Colorectal Neoplasms/genetics , DNA Methylation/drug effects , Epigenesis, Genetic/drug effects , Female , Humans , Irinotecan/administration & dosage , Irinotecan/pharmacokinetics , Long Interspersed Nucleotide Elements/genetics , Male , Middle Aged , Neoplasm Metastasis , Neoplasm Staging , Retreatment , Treatment OutcomeABSTRACT
Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. In the metastatic setting, the majority of patients respond to initial therapies but eventually develop resistance and progress. In this study, we test the hypothesis that priming with epigenetic therapy sensitizes CRC cell lines, which were previously resistant to subsequent chemotherapeutic agents. When multiple CRC cell lines are first exposed to 500 nM of the DNA demethylating agent, 5-aza-cytidine (AZA) in-vitro, and the cells then established as in-vivo xenografts in untreated NOD-SCID mice; there is an enhanced response to cytotoxic chemotherapy with agents commonly used in CRC treatment. For irinotecan (IRI), growth diminished by 16-62 fold as assessed, by both proliferation (IC50) and anchorage independent cell growth soft agar assays. Treatment of resistant HCT116 cell line along with in-vivo, for CRC line xenografts, AZA plus IRI again exhibits this synergistic response with significant improvement in survival and tumor regression in the mice. Genome-wide expression correlates changes in pathways for cell adhesion and DNA repair with the above responses. A Phase 1/2 clinical trial testing this concept is already underway testing the clinical efficacy of this concept in IRI resistant, metastatic CRC (NCT01896856).
Subject(s)
Antineoplastic Agents/therapeutic use , Azacitidine/therapeutic use , Camptothecin/analogs & derivatives , Colorectal Neoplasms/drug therapy , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Animals , Antineoplastic Agents/toxicity , Azacitidine/toxicity , Caco-2 Cells , Camptothecin/therapeutic use , Camptothecin/toxicity , Cell Adhesion/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , DNA Methylation/drug effects , DNA Repair/drug effects , Gene Expression/drug effects , Gene Expression Profiling , HCT116 Cells , Humans , Irinotecan , Long Interspersed Nucleotide Elements/genetics , Mice , Mice, Inbred NOD , Mice, SCIDABSTRACT
We previously developed a novel tumor subtype classification model for duodenal adenocarcinomas based on a combination of the CpG island methylator phenotype (CIMP) and MLH1 methylation status. Here, we tested the prognostic value of this model in stage II colorectal cancer (CRC) patients. Tumors were assigned to CIMP+/MLH1-unmethylated (MLH1-U), CIMP+/MLH1-methylated (MLH1-M), CIMP-/MLH1-U, or CIMP-/MLH1-M groups. Age, tumor location, lymphovascular invasion, and mucin production differed among the four patient subgroups, and CIMP+/MLH1-U tumors were more likely to have lymphovascular invasion and mucin production. Kaplan-Meier analyses revealed differences in both disease-free survival (DFS) and overall survival (OS) among the four groups. In a multivariate analysis, CIMP/MLH1 methylation status was predictive of both DFS and OS, and DFS and OS were shortest in CIMP+/MLH1-U stage II CRC patients. These results suggest that tumor subtype classification based on the combination of CIMP and MLH1 methylation status is informative in stage II CRC patients, and that CIMP+/MLH1-U tumors exhibit aggressive features and are associated with poor clinical outcomes.
Subject(s)
Colorectal Neoplasms/genetics , CpG Islands , DNA Methylation , MutL Protein Homolog 1/genetics , Adult , Aged , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Female , Humans , Male , Middle Aged , Neoplasm Staging , Phenotype , PrognosisABSTRACT
Epigenetics is a relatively recent field of molecular biology that has arisen over the past 25 years. Cancer is now understood to be a disease of widespread epigenetic dysregulation that interacts extensively with underlying genetic mutations. The development of drugs targeting these processes has rapidly progressed; with several drugs already FDA approved as first-line therapy in hematological malignancies. Gastrointestinal (GI) cancers possess high degrees of epigenetic dysregulation, exemplified by subtypes such as CpG island methylator phenotype (CIMP), and the potential benefit of epigenetic therapy in these cancers is evident. The application of epigenetic drugs in solid tumors, including GI cancers, is just emerging, with increased understanding of the cancer epigenome. In this review, we provide a brief overview of cancer epigenetics and the epigenetic targets of therapy including deoxyribonucleic acid (DNA) methylation, histone modifications, and chromatin remodeling. We discuss the epigenetic drugs currently in use, with a focus on DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, and explain the pharmacokinetic and mechanistic challenges in their application. We present the strategies employed in incorporating these drugs into the treatment of GI cancers, and explain the concept of the cancer stem cell in epigenetic reprogramming and reversal of chemo resistance. We discuss the most promising combination strategies in GI cancers including: (1) epigenetic sensitization to radiotherapy, (2) epigenetic sensitization to cytotoxic chemotherapy, and (3) epigenetic immune modulation and priming for immune therapy. Finally, we present preclinical and clinical trial data employing these strategies thus far in various GI cancers including colorectal, esophageal, gastric, and pancreatic cancer.
ABSTRACT
Tumor necrosis factor-alpha (TNF-α), a member of the TNF superfamily, was the first cytokine to be evaluated for cancer biotherapy. However, the clinical use of TNF-α is severely limited by its toxicity. Currently, TNF-α is administered only through locoregional drug delivery systems such as isolated limb perfusion and isolated hepatic perfusion. To reduce the systemic toxicity of TNF-α, various strategies have been explored over the last several decades. This review summarizes current state-of-the-art targeted cancer therapy using TNF-α. Passive targeting, cell-based therapy, gene therapy with inducible or tissue-specific promoters, targeted polymer-DNA complexes, tumor pre-targeting, antibody-TNF-α conjugate, scFv/TNF-α fusion proteins, and peptide/TNF-α fusion proteins have all been investigated to combat cancer. Many of these agents are already in advanced clinical trials. Molecular imaging, which can significantly speed up the drug development process, and nanomedicine, which can integrate both imaging and therapeutic components, has the potential to revolutionize future cancer patient management. Cooperative efforts from scientists within multiple disciplines, as well as close partnerships among many organizations/entities, are needed to quickly translate novel TNF-α-based therapeutics into clinical investigation.