ABSTRACT
Leucine-rich repeat-containing, G protein-coupled receptor 5 (LGR5) is highly expressed in colorectal cancer and cancer stem cells (CSCs) that play important roles in tumor initiation, progression, and metastasis. Loss of LGR5 has been shown to enhance therapy resistance. However, the molecular mechanisms that mediate this resistance remain elusive. In this study, we demonstrate conversion of LGR5+ colorectal cancer cells to an LGR5- state in response to chemotherapy, LGR5- targeted antibody-drug conjugates (ADCs), or LGR5 gene ablation led to activation of STAT3. Further investigation revealed increased STAT3 activation occurred as a result of increased mesenchymal epithelial transition (MET) factor receptor activity. LGR5 overexpression decreased MET-STAT3 activity and sensitized colorectal cancer cells to therapy. STAT3 inhibition suppressed MET phosphorylation, while constitutively active STAT3 reduced LGR5 levels and increased MET activity, suggesting a potential feedback mechanism. Combination treatment of MET-STAT3 inhibitors with irinotecan or antibody-drug conjugates (ADCs) substantiated synergistic effects in colorectal cancer cells and tumor organoids. In colorectal cancer xenografts, STAT3 inhibition combined with irinotecan enhanced tumor growth suppression and prolonged survival. These findings suggest a mechanism by which drug-resistant LGR5- colorectal cancer cells acquire a survival advantage through activation of MET-STAT3 and provide rationale for new treatment strategies to target colorectal cancer.
Subject(s)
Colorectal Neoplasms , Immunoconjugates , Humans , Down-Regulation , Irinotecan/pharmacology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Cell Line, Tumor , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Neoplastic Stem Cells/metabolism , Immunoconjugates/pharmacology , STAT3 Transcription Factor/geneticsABSTRACT
BACKGROUND: Long-term prognosis remains poor for colorectal cancer (CRC) patients with advanced disease due to treatment resistance. The identification of novel targets is essential for the development of new therapeutic approaches. GPR56, an adhesion GPCR, is highly expressed in CRC tumours and correlates with poor survival. Here, we describe the generation and preclinical evaluation of a novel ADC consisting of an anti-GPR56 antibody (10C7) conjugated with the DNA-damaging payload duocarmycin. METHODS: RNA-seq dataset analysis was performed to determine GPR56 expression in CRC subtypes. The specificity of binding, epitope mapping, and internalisation of 10C7 was examined. 10C7 was conjugated to payload and ADC cytotoxicity was assessed against a panel of CRC cell lines and tumour organoids. Antitumour efficacy was evaluated in xenograft models of CRC cell lines and patient-derived tumours. RESULTS: High GPR56 was shown to be associated with the microsatellite stable (MSS) subtype that accounts for 80-85% of CRC. GPR56 ADC selectively induced cytotoxicity in CRC cells and tumour organoids at low nanomolar potency in a GPR56-dependent manner and showed significant antitumour efficacy against GPR56-expressing xenograft models. CONCLUSIONS: This study provides the rationale for the future development of a GPR56-targeted ADC approach to potentially treat a large fraction of MSS CRC patients.
Subject(s)
Colorectal Neoplasms , Immunoconjugates , Humans , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Immunoconjugates/pharmacology , Immunoconjugates/therapeutic use , Prognosis , Receptors, G-Protein-Coupled/metabolismABSTRACT
GPR56 is a member of the adhesion G-protein-coupled receptor family shown to play important roles in cell adhesion, brain development, immune function, and tumorigenesis. GPR56 is highly upregulated in colorectal cancer and correlates with poor prognosis. Several studies have shown GPR56 couples to the Gα12/13 class of heterotrimeric G-proteins to promote RhoA activation. However, due to its structural complexity and lack of a high-affinity receptor-specific ligand, the complete GPR56 signaling mechanism remains largely unknown. To delineate the activation mechanism and intracellular signaling functions of GPR56, we generated a monoclonal antibody (mAb) that binds with high affinity and specificity to the extracellular domain (ECD). Using deletion mutants, we mapped the mAb binding site to the GAIN domain, which mediates membrane-proximal autoproteolytic cleavage of the ECD. We showed that GPR56 overexpression in 293T cells leads to increased phosphorylation of Src, Fak, and paxillin adhesion proteins and activation of the Gα12/13-RhoA-mediated serum response factor (SRF) pathway. Treatment with the mAb potentiated Src-Fak phosphorylation, RhoA-SRF signaling, and cell adhesion. Consistently, GPR56 knockdown in colorectal cancer cells decreased Src-Fak pathway phosphorylation and cell adhesion. Interestingly, GPR56-mediated activation of Src-Fak phosphorylation occurred independent of RhoA, yet mAb-induced potentiation of RhoA-SRF signaling was Src-dependent. Furthermore, we show that the C-terminal portion of the Serine-Threonine-Proline-rich (STP) region, adjacent to the GAIN domain, was required for Src-Fak activation. However, autoproteolytic cleavage of the ECD was dispensable. These data support a new ECD-dependent mechanism by which GPR56 functions to regulate adhesion through activation of Src-Fak signaling.
Subject(s)
Colorectal Neoplasms/genetics , Focal Adhesion Kinase 1/genetics , Receptors, G-Protein-Coupled/genetics , Serum Response Factor/genetics , rhoA GTP-Binding Protein/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Carcinogenesis/genetics , Cell Adhesion/immunology , Cell Line, Tumor , Colorectal Neoplasms/immunology , Colorectal Neoplasms/pathology , Focal Adhesion Kinase 1/immunology , Gene Expression Regulation, Neoplastic/genetics , Humans , Paxillin/genetics , Paxillin/immunology , Phosphorylation/genetics , Receptors, G-Protein-Coupled/immunology , Serum Response Factor/immunology , Signal Transduction/genetics , rhoA GTP-Binding Protein/immunology , src-Family Kinases/genetics , src-Family Kinases/immunologyABSTRACT
Enterococcus faecalis is a significant cause of hospital-acquired bacteremia. Herein, the discovery is reported that cardiac microlesions form during severe bacteremic E. faecalis infection in mice. The cardiac microlesions were identical in appearance to those formed by Streptococcus pneumoniae during invasive pneumococcal disease. However, E. faecalis does not encode the virulence determinants implicated in pneumococcal microlesion formation. Rather, disulfide bond forming protein A (DsbA) was found to be required for E. faecalis virulence in a Caenorhabditis elegans model and was necessary for efficient cardiac microlesion formation. Furthermore, E. faecalis promoted cardiomyocyte apoptotic and necroptotic cell death at sites of microlesion formation. Additionally, loss of DsbA caused an increase in proinflammatory cytokines, unlike the wild-type strain, which suppressed the immune response. In conclusion, we establish that E. faecalis is capable of forming cardiac microlesions and identify features of both the bacterium and the host response that are mechanistically involved.
Subject(s)
Bacteremia/microbiology , Bacteremia/pathology , Enterococcus faecalis/pathogenicity , Heart Diseases/microbiology , Heart Diseases/pathology , Heart , Animals , Apoptosis , Bacterial Proteins/metabolism , Caenorhabditis elegans/microbiology , Cell Death , Cytokines , Disease Models, Animal , Gram-Positive Bacterial Infections/microbiology , Gram-Positive Bacterial Infections/pathology , Mice , Necroptosis , Pneumococcal Infections/microbiology , Streptococcus pneumoniae/pathogenicity , Thioredoxins , Virulence , Virulence FactorsABSTRACT
Gall-forming arthropods are highly specialized herbivores that, in combination with their hosts, produce extended phenotypes with unique morphologies [1]. Many are economically important, and others have improved our understanding of ecology and adaptive radiation [2]. However, the mechanisms that these arthropods use to induce plant galls are poorly understood. We sequenced the genome of the Hessian fly (Mayetiola destructor; Diptera: Cecidomyiidae), a plant parasitic gall midge and a pest of wheat (Triticum spp.), with the aim of identifying genic modifications that contribute to its plant-parasitic lifestyle. Among several adaptive modifications, we discovered an expansive reservoir of potential effector proteins. Nearly 5% of the 20,163 predicted gene models matched putative effector gene transcripts present in the M. destructor larval salivary gland. Another 466 putative effectors were discovered among the genes that have no sequence similarities in other organisms. The largest known arthropod gene family (family SSGP-71) was also discovered within the effector reservoir. SSGP-71 proteins lack sequence homologies to other proteins, but their structures resemble both ubiquitin E3 ligases in plants and E3-ligase-mimicking effectors in plant pathogenic bacteria. SSGP-71 proteins and wheat Skp proteins interact in vivo. Mutations in different SSGP-71 genes avoid the effector-triggered immunity that is directed by the wheat resistance genes H6 and H9. Results point to effectors as the agents responsible for arthropod-induced plant gall formation.
Subject(s)
Chromosomes/genetics , Diptera/genetics , Multigene Family/genetics , Phylogeny , Plant Tumors/genetics , Triticum/parasitology , Adaptation, Biological/genetics , Amino Acid Sequence , Animals , Base Sequence , Diptera/metabolism , Larva/metabolism , Models, Genetic , Molecular Sequence Data , Sequence Analysis, DNA , Sequence Homology , Sexual Behavior, Animal/physiology , Two-Hybrid System Techniques , Ubiquitin-Protein Ligases/geneticsABSTRACT
Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.
Subject(s)
Arthropods/genetics , Genome , Synteny , Animals , Circadian Rhythm Signaling Peptides and Proteins/genetics , DNA Methylation , Evolution, Molecular , Female , Genome, Mitochondrial , Hormones/genetics , Male , Multigene Family , Phylogeny , Polymorphism, Genetic , Protein Kinases/genetics , RNA, Untranslated/genetics , Receptors, Odorant/genetics , Selenoproteins/genetics , Sex Chromosomes , Transcription Factors/geneticsABSTRACT
BACKGROUND: Hyperthermia (HS) is a well-studied teratogen that induces serious malformations, including neural tube defects. Our previous studies have shown that HS induces apoptosis by activating the mitochondrial apoptotic pathway. Prior to activation of the mitochondrial apoptotic pathway, HS also activates p53 and its target genes. In the present study, we determine whether p53 and/or p21 play a role as teratogen suppressors or inducers of HS-induced malformations. METHODS: Pregnant mice carrying all three p53 or p21 genotype embryos were exposed to HS on day 8.5. Subsequently, fetuses were collected on day 15.5, and genotyped. In addition to genotype, we also determined the number of resorptions and dead fetuses as well as the number and types of external malformations. RESULTS: In the absence of HS exposure, fetuses exhibiting exencephaly and spina bifida were observed in approximately 11% of p53 -/- fetuses, whereas no malformations were observed among p21 -/- fetuses. Exposure to HS resulted in an increase in exencephaly and polydactyly in fetuses of all three p53 genotypes. However, the incidence of these malformations was statistically significantly higher in p53 -/- compared to p53 +/- and p53 +/+ fetuses. Exencephaly was the only malformation observed in p21 fetuses exposed to HS, with an approximately 2-fold increase among p21 +/- and a 3-fold increase among p21 -/- compared to p21 +/+ fetuses. CONCLUSIONS: Our study confirms that p53 plays a role in normal development and has shown, for the first time that p53 and p21 function to suppress HS-induced malformations.