Inhibition of EGFR/ErbB does not protect against C. difficile toxin B.

Clostridioides difficile is a common cause of diarrhea and mortality, especially in immunosuppressed and hospitalized patients. C. difficile is a toxin-mediated disease, but the host cell receptors for C. difficile toxin B (TcdB) have only recently been revealed. Emerging data suggest TcdB interacts with receptor tyrosine kinases during infection. In particular, TcdB can elicit Epidermal Growth Factor Receptor (EGFR) transactivation in human colonic epithelial cells. The mechanisms for this function are not well understood, and the involvement of other receptors in the EGFR family of Erythroblastic Leukemia Viral Oncogene Homolog (ErbB) receptors remains unclear. Furthermore, in an siRNA-knockdown screen for protective genes involved with TcdB toxin pathogenesis, we show ErbB2 and ErbB3 loss resulted in increased cell viability. We hypothesize TcdB induces the transactivation of EGFR and/or ErbB receptors as a component of its cell-killing mechanism. Here, we show in vivo intrarectal instillation of TcdB in mice leads to phosphorylation of ErbB2 and ErbB3. However, immunohistochemical staining for phosphorylated ErbB2 and ErbB3 indicated no discernible difference between control and TcdB-treated mice for epithelial phospho-ErbB2 and phospho-ErbB3. Human colon cancer cell lines (HT29, Caco-2) exposed to TcdB were not protected by pre-treatment with lapatinib, an EGFR/ErbB2 inhibitor. Similarly, lapatinib pre-treatment failed to protect normal human colonoids from TcdB-induced cell death. Neutralizing antibodies against mouse EGFR failed to protect mice from TcdB intrarectal instillation as measured by edema, inflammatory infiltration, and epithelial injury. Our findings suggest TcdB-induced colonocyte cell death does not require EGFR/ErbB receptor tyrosine kinase activation.

Molecular cartography uncovers evolutionary and microenvironmental dynamics in sporadic colorectal tumors.

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Analysis of spatial multi-omic data from 31 human colorectal specimens enabled phylogeographic mapping of tumor evolution that revealed individualized progression trajectories and accompanying microenvironmental and clonal alterations. Phylogeographic mapping ordered genetic events, classified tumors by their evolutionary dynamics, and placed clonal regions along global pseudotemporal progression trajectories encompassing the chromosomal instability (CIN+) and hypermutated (HM) pathways. Integrated single-cell and spatial transcriptomic data revealed recurring epithelial programs and infiltrating immune states along progression pseudotime. We discovered an immune exclusion signature (IEX), consisting of extracellular matrix regulators DDR1, TGFBI, PAK4, and DPEP1, that charts with CIN+ tumor progression, is associated with reduced cytotoxic cell infiltration, and shows prognostic value in independent cohorts. This spatial multi-omic atlas provides insights into colorectal tumor-microenvironment co-evolution, serving as a resource for stratification and targeted treatments.

Distinct roles for interleukin-23 receptor signaling in regulatory T cells in sporadic and inflammation-associated carcinogenesis.

Introduction: The pro-inflammatory cytokine interleukin-23 (IL-23) has been implicated in colorectal cancer (CRC). Yet, the cell-specific contributions of IL-23 receptor (IL-23R) signaling in CRC remain unknown. One of the cell types that highly expresses IL-23R are colonic regulatory T cells (Treg cells). The aim of this study was to define the contribution of Treg cell-specific IL-23R signaling in sporadic and inflammation-associated CRC. Methods: In mice, the role of IL-23R in Treg cells in colitis-associated cancer (CAC) was investigated using azoxymethane/dextran sodium sulphate in wild-type Treg cell reporter mice (WT, Foxp3 YFP-iCre), and mice harboring a Treg cell-specific deletion of IL-23 (Il23r ΔTreg). The role of IL-23R signaling in Treg cells in sporadic CRC was examined utilizing orthotopic injection of the syngeneic colon cancer cell line MC-38 submucosally into the colon/rectum of mice. The function of macrophages was studied using clodronate. Finally, single-cell RNA-seq of a previously published dataset in human sporadic cancer was reanalyzed to corroborate these findings. Results: In CAC, Il23r ΔTreg mice had increased tumor size and increased dysplasia compared to WT mice that was associated with decreased tumor-infiltrating macrophages. In the sporadic cancer model, Il23r ΔTreg mice had increased survival and decreased tumor size compared to WT mice. Additionally, MC-38 tumors of Il23r ΔTreg mice exhibited a higher frequency of pro-inflammatory macrophages and IL-17 producing CD4+ T cells. The decreased tumor size in Il23r ΔTreg mice was macrophage-dependent. These data suggest that loss of IL-23R signaling in Treg cells permits IL-17 production by CD4+ T cells that in turn promotes pro-inflammatory macrophages to clear tumors. Finally, analysis of TCGA data and single-cell RNA-seq analysis of a previously published dataset in human sporadic cancer, revealed that IL23R was highly expressed in CRC compared to other cancers and specifically in tumor-associated Treg cells. Conclusion: Inflammation in colorectal carcinogenesis differs with respect to the contribution of IL-23R signaling in regulatory T cells.

Human Colon Cancer-Derived Clostridioides difficile Strains Drive Colonic Tumorigenesis in Mice.

Defining the complex role of the microbiome in colorectal cancer and the discovery of novel, protumorigenic microbes are areas of active investigation. In the present study, culturing and reassociation experiments revealed that toxigenic strains of Clostridioides difficile drove the tumorigenic phenotype of a subset of colorectal cancer patient-derived mucosal slurries in germ-free ApcMin/+ mice. Tumorigenesis was dependent on the C. difficile toxin TcdB and was associated with induction of Wnt signaling, reactive oxygen species, and protumorigenic mucosal immune responses marked by the infiltration of activated myeloid cells and IL17-producing lymphoid and innate lymphoid cell subsets. These findings suggest that chronic colonization with toxigenic C. difficile is a potential driver of colorectal cancer in patients. SIGNIFICANCE: Colorectal cancer is a leading cause of cancer and cancer-related deaths worldwide, with a multifactorial etiology that likely includes procarcinogenic bacteria. Using human colon cancer specimens, culturing, and murine models, we demonstrate that chronic infection with the enteric pathogen C. difficile is a previously unrecognized contributor to colonic tumorigenesis. See related commentary by Jain and Dudeja, p. 1838. This article is highlighted in the In This Issue feature, p. 1825.

Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps.

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.

Colon epithelial cell TGFß signaling modulates the expression of tight junction proteins and barrier function in mice.

Defective barrier function is a predisposing factor in inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Although TGFß signaling defects have been associated with IBD and CAC, few studies have examined the relationship between TGFß and intestinal barrier function. Here, we examine the role of TGFß signaling via SMAD4 in modulation of colon barrier function. The Smad4 gene was conditionally deleted in the intestines of adult mice and intestinal permeability assessed using an in vivo 4 kDa FITC-Dextran (FD4) permeability assay. Mouse colon was isolated for gene expression (RNA-sequencing), Western blot, and immunofluorescence analysis. In vitro colon organoid culture was utilized to assess junction-related gene expression by qPCR and transepithelial resistance (TER). In silico analyses of human IBD and colon cancer databases were performed. Mice lacking intestinal expression of Smad4 demonstrate increased colonic permeability to FD4 without gross mucosal damage. mRNA/protein expression analyses demonstrate significant increases in Cldn2/Claudin 2 and Cldn8/Claudin 8, and decreases in Cldn3, Cldn4, and Cldn7/Claudin 7 with intestinal SMAD4 loss in vivo without changes in Claudin protein localization. TGFß1/BMP2 treatment of polarized SMAD4+ colonoids increases TER. Cldn2, Cldn4, Cldn7, and Cldn8 are regulated by canonical TGFß signaling, and TGFß-dependent regulation of these genes is dependent on nascent RNA transcription (Cldn2, Cldn4, Cldn8) but not nascent protein translation (Cldn4, Cldn8). Human IBD/colon cancer specimens demonstrate decreased SMAD4, CLDN4, CLDN7, and CLDN8 and increased CLDN2 compared with healthy controls. Canonical TGFß signaling modulates the expression of tight junction proteins and barrier function in mouse colon.NEW & NOTEWORTHY We demonstrate that canonical TGFß family signaling modulates the expression of critical tight junction proteins in colon epithelial cells, and that expression of these tight junction proteins is associated with maintenance of colon epithelial barrier function in mice.

Protein kinase A-mediated phosphorylation of naked cuticle homolog 2 stimulates cell-surface delivery of transforming growth factor-α for epidermal growth factor receptor transactivation.

The classic mode of G protein-coupled receptor (GPCR)-mediated transactivation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR) transactivation occurs via matrix metalloprotease (MMP)-mediated cleavage of plasma membrane-anchored EGFR ligands. Herein, we show that the Gαs-activating GPCR ligands vasoactive intestinal peptide (VIP) and prostaglandin E2 (PGE2 ) transactivate EGFR through increased cell-surface delivery of the EGFR ligand transforming growth factor-α (TGFα) in polarizing madin-darby canine kidney (MDCK) and Caco-2 cells. This is achieved by PKA-mediated phosphorylation of naked cuticle homolog 2 (NKD2), previously shown to bind TGFα and direct delivery of TGFα-containing vesicles to the basolateral surface of polarized epithelial cells. VIP and PGE2 rapidly activate protein kinase A (PKA) that then phosphorylates NKD2 at Ser-223, a process that is facilitated by the molecular scaffold A-kinase anchoring protein 12 (AKAP12). This phosphorylation stabilized NKD2, ensuring efficient cell-surface delivery of TGFα and increased EGFR activation. Thus, GPCR-triggered, PKA/AKAP12/NKD2-regulated targeting of TGFα to the cell surface represents a new mode of EGFR transactivation that occurs proximal to ligand cleavage by MMPs.

p120-catenin controls contractility along the vertical axis of epithelial lateral membranes.

In vertebrate epithelia, p120-catenin (hereafter referred to as p120; also known as CTNND1) mediates E-cadherin stability and suppression of RhoA. Genetic ablation of p120 in various epithelial tissues typically causes striking alterations in tissue function and morphology. Although these effects could very well involve p120's activity towards Rho, ascertaining the impact of this relationship has been complicated by the fact that p120 is also required for cell-cell adhesion. Here, we have molecularly uncoupled p120's cadherin-stabilizing and RhoA-suppressing activites. Unexpectedly, removing p120's Rho-suppressing activity dramatically disrupted the integrity of the apical surface, irrespective of E-cadherin stability. The physical defect was tracked to excessive actomyosin contractility along the vertical axis of lateral membranes. Thus, we suggest that p120's distinct activities towards E-cadherin and Rho are molecularly and functionally coupled and this, in turn, enables the maintenance of cell shape in the larger context of an epithelial monolayer. Importantly, local suppression of contractility by cadherin-bound p120 appears to go beyond regulating cell shape, as loss of this activity also leads to major defects in epithelial lumenogenesis.

DIPA-family coiled-coils bind conserved isoform-specific head domain of p120-catenin family: potential roles in hydrocephalus and heterotopia.

p120-catenin (p120) modulates adherens junction (AJ) dynamics by controlling the stability of classical cadherins. Among all p120 isoforms, p120-3A and p120-1A are the most prevalent. Both stabilize cadherins, but p120-3A is preferred in epithelia, whereas p120-1A takes precedence in neurons, fibroblasts, and macrophages. During epithelial-to-mesenchymal transition, E- to N-cadherin switching coincides with p120-3A to -1A alternative splicing. These isoforms differ by a 101-amino acid "head domain" comprising the p120-1A N-terminus. Although its exact role is unknown, the head domain likely mediates developmental and cancer-associated events linked to p120-1A expression (e.g., motility, invasion, metastasis). Here we identified delta-interacting protein A (DIPA) as the first head domain-specific binding partner and candidate mediator of isoform 1A activity. DIPA colocalizes with AJs in a p120-1A- but not 3A-dependent manner. Moreover, all DIPA family members (Ccdc85a, Ccdc85b/DIPA, and Ccdc85c) interact reciprocally with p120 family members (p120, δ-catenin, p0071, and ARVCF), suggesting significant functional overlap. During zebrafish neural tube development, both knockdown and overexpression of DIPA phenocopy N-cadherin mutations, an effect bearing functional ties to a reported mouse hydrocephalus phenotype associated with Ccdc85c. These studies identify a novel, highly conserved interaction between two protein families that may participate either individually or collectively in N-cadherin-mediated development.