Bottom-up or Top-down: Inflammation Reprograms Paneth Cells to Develop Bowel Cancers.

The origins of colorectal cancer have long been a subject of intense debate. Early observations noted cancer formation in the human gut slightly above the base of crypts, the structural and functional units of the regenerative compartment of the intestinal epithelium. This suggested that the cells of origin for colorectal cancer reside close to the crypt-villus junction, where more differentiated cells are located. However, the specific induction of early cancer-initiating mutations within differentiated cells failed to initiate cancer. The subsequent identification of long-lived Lgr5+ intestinal stem cells and investigations into their role in cancer development further shifted the earlier views, leading to the widely accepted theory that colorectal cancer arises from stem cells and progenitors located at the base of crypts. A recent study published in Nature Genetics by Mathijs P. Verhagen and colleagues challenges this paradigm, providing compelling evidence that differentiated nonstem cell lineages, particularly Paneth cells, can serve as a source of intestinal tumorigenesis, especially in the context of inflammation and the consumption of a Western-style diet. This work significantly advances our understanding of the colorectal cancer initiation process and provides a new paradigm that may explain the increasingly higher incidence of colorectal cancer in younger people.

Bcl-6 expression by CD4+ T cells determines concomitant immunity and host resistance across distinct parasitic infections.

Cluster of differentiation (CD4+) T cells consist of multiple subtypes, defined by expression of lineage-specific transcription factors, that contribute to the control of infectious diseases by providing help to immune and nonimmune target cells. In the current study, we examined the role of B cell lymphoma (Bcl)-6, a transcriptional repressor and master regulator of T follicular helper cell differentiation, in T cell-mediated host defense against intestinal and systemic parasitic infections. We demonstrate that while Bcl-6 expression by CD4+ T cells is critical for antibody-mediated protective immunity against secondary infection with the nematode Heligmosoides polygyrus bakeri, it paradoxically compromises worm expulsion during primary infection by limiting the generation of interleukin-10 (IL-10)-producing Gata3+ T helper 2 cells. Enhanced worm expulsion in the absence of Bcl-6 expressing T cells was associated with amplified intestinal goblet cell differentiation and increased generation of alternatively activated macrophages, effects that were reversed by neutralization of IL-10 signals. An increase in IL-10 production by Bcl-6-deficient CD4+ T cells was also evident in the context of systemic Leishmania donovani infection, but in contrast to Heligmosoides polygyrus bakeri infection, compromised T helper 1-mediated liver macrophage activation and increased susceptibility to this distinct parasitic challenge. Collectively, our studies suggest that host defense pathways that protect against parasite superinfection and lethal systemic protozoal infections can be engaged at the cost of compromised primary resistance to well-tolerated helminths.

Multiplex In Situ Hybridization in the Study of Acute Kidney Injury : Multiplex In Situ Hybridization in AKI.

Recently developed in situ hybridization (ISH) methods, such as RNAscope™, have greatly expanded the accessibility and usefulness of ISH in biomedical research. Among many other advantages over traditional ISH, these newer methods enable the simultaneous use of multiple probes, including combination with antibody or lectin staining. We herein illustrate the application of RNAscope™ multiplex ISH in the study of the adapter protein Dok-4 in acute kidney injury (AKI). Specifically, we used multiplex ISH to define the expression of Dok-4 and some of its putative binding partners, together with nephron segment markers, as well as markers of proliferation and tubular injury. We also illustrate the use of QuPath image analysis software to perform quantitative analyses of multiplex ISH. Furthermore, we describe how these analyses can exploit the uncoupling of mRNA and protein expression in a knockout (KO) mouse created by CRISPR/CAS9-mediated frame shift to carry out highly focused molecular phenotyping studies at the single-cell level.

Dangerous liaisons: how helminths manipulate the intestinal epithelium.

Intestinal helminths remain highly pervasive throughout the animal kingdom by modulating multiple aspects of the host immune response. The intestinal epithelium functions as a physical barrier as well as a sentinel innate immune tissue with the ability to sense and respond to infectious agents. Although helminths form intimate interactions with the epithelium, comprehensive knowledge about host-helminth interactions at this dynamic interface is lacking. In addition, little is known about the ability of helminths to directly shape the fate of this barrier tissue. Here, we review the diverse pathways by which helminths regulate the epithelium and highlight the emerging field of direct helminth regulation of intestinal stem cell (ISC) fate and function.

Helminth-induced reprogramming of the stem cell compartment inhibits type 2 immunity.

Enteric helminths form intimate physical connections with the intestinal epithelium, yet their ability to directly alter epithelial stem cell fate has not been resolved. Here we demonstrate that infection of mice with the parasite Heligmosomoides polygyrus bakeri (Hpb) reprograms the intestinal epithelium into a fetal-like state marked by the emergence of Clusterin-expressing revival stem cells (revSCs). Organoid-based studies using parasite-derived excretory-secretory products reveal that Hpb-mediated revSC generation occurs independently of host-derived immune signals and inhibits type 2 cytokine-driven differentiation of secretory epithelial lineages that promote their expulsion. Reciprocally, type 2 cytokine signals limit revSC differentiation and, consequently, Hpb fitness, indicating that helminths compete with their host for control of the intestinal stem cell compartment to promote continuation of their life cycle.

An integrated model of acinar to ductal metaplasia-related N7-methyladenosine regulators predicts prognosis and immunotherapy in pancreatic carcinoma based on digital spatial profiling.

Acinar-to-ductal metaplasia (ADM) is a recently recognized, yet less well-studied, precursor lesion of pancreatic ductal adenocarcinoma (PDAC) developed in the setting of chronic pancreatitis. Through digital spatial mRNA profiling, we compared ADM and adjacent PDAC tissues from patient samples to unveil the bridging genes during the malignant transformation of pancreatitis. By comparing the bridging genes with the 7-methylguanosine (m7G)-seq dataset, we screened 19 m7G methylation genes for a subsequent large sample analysis. We constructed the "m7G score" model based on the RNA-seq data for pancreatic cancer in The Cancer Genome Atlas (TCGA) database and The Gene Expression Omnibus (GEO) database. Tumors with a high m7G score were characterized by increased immune cell infiltration, increased genomic instability, higher response rate to combined immune checkpoint inhibitors (ICIs), and overall poor survival. These findings indicate that the m7G score is associated with tumor invasiveness, immune cell infiltration, ICI treatment response, and overall patients' survival. We also identified FN1 and ITGB1 as core genes in the m7Gscore model, which affect immune cell infiltration and genomic instability not only in pancreatic cancer but also in pan-cancer. FN1 and ITGB1 can inhibit immune T cell activition by upregulation of macrophages and neutrophils, thereby leading to immune escape of pancreatic cancer cells and reducing the response rate of ICI treatment.

NUAK1 promotes organ fibrosis via YAP and TGF-ß/SMAD signaling.

Fibrosis is a central pathway that drives progression of multiple chronic diseases, yet few safe and effective clinical antifibrotic therapies exist. In most fibrotic disorders, transforming growth factor-ß (TGF-ß)-driven scarring is an important pathologic feature and a key contributor to disease progression. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are two closely related transcription cofactors that are important for coordinating fibrogenesis after organ injury, but how they are activated in response to tissue injury has, so far, remained unclear. Here, we describe NUAK family kinase 1 (NUAK1) as a TGF-ß-inducible profibrotic kinase that is up-regulated in multiple fibrotic organs in mice and humans. Mechanistically, we show that TGF-ß induces a rapid increase in NUAK1 in fibroblasts. NUAK1, in turn, can promote profibrotic YAP and TGF-ß/SMAD signaling, ultimately leading to organ scarring. Moreover, activated YAP and TAZ can induce further NUAK1 expression, creating a profibrotic positive feedback loop that enables persistent fibrosis. Using mouse models of kidney, lung, and liver fibrosis, we demonstrate that this fibrogenic signaling loop can be interrupted via fibroblast-specific loss of NUAK1 expression, leading to marked attenuation of fibrosis. Pharmacologic NUAK1 inhibition also reduced scarring, either when initiated immediately after injury or when initiated after fibrosis was already established. Together, our data suggest that NUAK1 plays a critical, previously unrecognized role in fibrogenesis and represents an attractive target for strategies that aim to slow fibrotic disease progression.

Taking a Step Back: Insights into the Mechanisms Regulating Gut Epithelial Dedifferentiation.

Despite the environmental constraints imposed upon the intestinal epithelium, this tissue must perform essential functions such as nutrient absorption and hormonal regulation, while also acting as a critical barrier to the outside world. These functions depend on a variety of specialized cell types that are constantly renewed by a rapidly proliferating population of intestinal stem cells (ISCs) residing at the base of the crypts of Lieberkühn. The niche components and signals regulating crypt morphogenesis and maintenance of homeostatic ISCs have been intensely studied over the last decades. Increasingly, however, researchers are turning their attention to unraveling the mechanisms driving gut epithelial regeneration due to physical damage or infection. It is now well established that injury to the gut barrier triggers major cell fate changes, demonstrating the highly plastic nature of the gut epithelium. In particular, lineage tracing and transcriptional profiling experiments have uncovered several injury-induced stem-cell populations and molecular markers of the regenerative state. Despite the progress achieved in recent years, several questions remain unresolved, particularly regarding the mechanisms driving dedifferentiation of the gut epithelium. In this review, we summarize the latest studies, primarily from murine models, that define the regenerative processes governing the gut epithelium and discuss areas that will require more in-depth investigation.

Binary pan-cancer classes with distinct vulnerabilities defined by pro- or anti-cancer YAP/TEAD activity.

Cancer heterogeneity impacts therapeutic response, driving efforts to discover over-arching rules that supersede variability. Here, we define pan-cancer binary classes based on distinct expression of YAP and YAP-responsive adhesion regulators. Combining informatics with in vivo and in vitro gain- and loss-of-function studies across multiple murine and human tumor types, we show that opposite pro- or anti-cancer YAP activity functionally defines binary YAPon or YAPoff cancer classes that express or silence YAP, respectively. YAPoff solid cancers are neural/neuroendocrine and frequently RB1-/-, such as retinoblastoma, small cell lung cancer, and neuroendocrine prostate cancer. YAP silencing is intrinsic to the cell of origin, or acquired with lineage switching and drug resistance. The binary cancer groups exhibit distinct YAP-dependent adhesive behavior and pharmaceutical vulnerabilities, underscoring clinical relevance. Mechanistically, distinct YAP/TEAD enhancers in YAPoff or YAPon cancers deploy anti-cancer integrin or pro-cancer proliferative programs, respectively. YAP is thus pivotal across cancer, but in opposite ways, with therapeutic implications.

REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway.

Persistent acinar to ductal metaplasia (ADM) is a recently recognized precursor of pancreatic ductal adenocarcinoma (PDAC). Here we show that the ADM area of human pancreas tissue adjacent to PDAC expresses significantly higher levels of regenerating protein 3A (REG3A). Exogenous REG3A and its mouse homolog REG3B induce ADM in the 3D culture of primary human and murine acinar cells, respectively. Both Reg3b transgenic mice and REG3B-treated mice with caerulein-induced pancreatitis develop and sustain ADM. Two out of five Reg3b transgenic mice with caerulein-induced pancreatitis show progression from ADM to pancreatic intraepithelial neoplasia (PanIN). Both in vitro and in vivo ADM models demonstrate activation of the RAS-RAF-MEK-ERK signaling pathway. Exostosin-like glycosyltransferase 3 (EXTL3) functions as the receptor for REG3B and mediates the activation of downstream signaling proteins. Our data indicates that REG3A/REG3B promotes persistent ADM through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway. Targeting REG3A/REG3B, its receptor EXTL3, or other downstream molecules could interrupt the ADM process and prevent early PDAC carcinogenesis.

Single-cell transcriptomes of the regenerating intestine reveal a revival stem cell.

The turnover of the intestinal epithelium is driven by multipotent LGR5+ crypt-base columnar cells (CBCs) located at the bottom of crypt zones1. However, CBCs are lost following injury, such as irradiation2, but the intestinal epithelium is nevertheless able to recover3. Thus, a second population of quiescent '+4' cells, or reserve stem cells (RSCs), has previously been proposed to regenerate the damaged intestine4-7. Although CBCs and RSCs were thought to be mutually exclusive4,8, subsequent studies have found that LGR5+ CBCs express RSC markers9 and that RSCs were dispensable-whereas LGR5+ cells were essential-for repair of the damaged intestine3. In addition, progenitors of absorptive enterocytes10, secretory cells11-15 and slow cycling LGR5+ cells16 have been shown to contribute to regeneration whereas the transcriptional regulator YAP1, which is important for intestinal regeneration, was suggested to induce a pro-survival phenotype in LGR5+ cells17. Thus, whether cellular plasticity or distinct cell populations are critical for intestinal regeneration remains unknown. Here we applied single-cell RNA sequencing to profile the regenerating mouse intestine and identified a distinct, damage-induced quiescent cell type that we term the revival stem cell (revSC). revSCs are marked by high clusterin expression and are extremely rare under homoeostatic conditions, yet give rise-in a temporal hierarchy-to all the major cell types of the intestine, including LGR5+ CBCs. After intestinal damage by irradiation, targeted ablation of LGR5+ CBCs, or treatment with dextran sodium sulfate, revSCs undergo a YAP1-dependent transient expansion, reconstitute the LGR5+ CBC compartment and are required to regenerate a functional intestine. These studies thus define a unique stem cell that is mobilized by damage to revive the homoeostatic stem cell compartment and regenerate the intestinal epithelium.

A feed forward loop enforces YAP/TAZ signaling during tumorigenesis.

In most solid tumors, the Hippo pathway is inactivated through poorly understood mechanisms that result in the activation of the transcriptional regulators, YAP and TAZ. Here, we identify NUAK2 as a YAP/TAZ activator that directly inhibits LATS-mediated phosphorylation of YAP/TAZ and show that NUAK2 induction by YAP/TAZ and AP-1 is required for robust YAP/TAZ signaling. Pharmacological inhibition or loss of NUAK2 reduces the growth of cultured cancer cells and mammary tumors in mice. Moreover, in human patient samples, we show that NUAK2 expression is elevated in aggressive, high-grade bladder cancer and strongly correlates with a YAP/TAZ gene signature. These findings identify a positive feed forward loop in the Hippo pathway that establishes a key role for NUAK2 in enforcing the tumor-promoting activities of YAP/TAZ. Our results thus introduce a new opportunity for cancer therapeutics by delineating NUAK2 as a potential target for re-engaging the Hippo pathway.

Use of Organoids to Characterize Signaling Pathways in Cancer Initiation.

The development of intestinal organoid technology has greatly accelerated research in the field of colorectal cancer. Contrary to traditional cancer cell lines, organoids are composed of multiple cell types arranged in 3D structures highly reminiscent of their native tissues. Thus, organoids provide a near-physiological and readily accessible model to study tissue morphogenesis, adult stem cell behavior and tumorigenesis. Here, we provide protocols for establishing intestinal organoid cultures from genetically modified mouse lines and describe methods to overexpress and knockout genes of interest using lentiviral-based approaches.

Hippo signalling in intestinal regeneration and cancer.

The Hippo pathway is a unique signalling module that regulates cell-specific transcriptional responses and responds to a wide range of intrinsic and extrinsic cues. Besides its classical role in restricting tissue size during development, Hippo signalling is now recognized to control numerous processes including cell proliferation, survival, cell fate determination, epithelial-to-mesenchymal transitions and cell migration. Because of its highly dynamic nature, the intestinal epithelium has served as an exceptional model to study the complex roles of Hippo signalling. In this review, we shall present an overview of Hippo function in the mammalian intestine and discuss the various mechanisms regulating Hippo signalling and how they contribute to intestinal regeneration and cancer.

A critical role for NF2 and the Hippo pathway in branching morphogenesis.

Branching morphogenesis is a complex biological process common to the development of most epithelial organs. Here we demonstrate that NF2, LATS1/2 and YAP play a critical role in branching morphogenesis in the mouse kidney. Removal of Nf2 or Lats1/2 from the ureteric bud (UB) lineage causes loss of branching morphogenesis that is rescued by loss of one copy of Yap and Taz, and phenocopied by YAP overexpression. Mosaic analysis demonstrates that cells with high YAP expression have reduced contribution to UB tips, similar to Ret(-/-) cells, and that YAP suppresses RET signalling and tip identity. Conversely, Yap/Taz UB-deletion leads to cyst-like branching and expansion of UB tip markers, suggesting a shift towards tip cell identity. Based on these data we propose that NF2 and the Hippo pathway locally repress YAP/TAZ activity in the UB to promote subsequent splitting of the tip to allow branching morphogenesis.

YAP and TAZ control peripheral myelination and the expression of laminin receptors in Schwann cells.

Myelination is essential for nervous system function. Schwann cells interact with neurons and the basal lamina to myelinate axons using known receptors, signals and transcription factors. In contrast, the transcriptional control of axonal sorting and the role of mechanotransduction in myelination are largely unknown. Yap and Taz are effectors of the Hippo pathway that integrate chemical and mechanical signals in cells. We describe a previously unknown role for the Hippo pathway in myelination. Using conditional mutagenesis in mice, we show that Taz is required in Schwann cells for radial sorting and myelination and that Yap is redundant with Taz. Yap and Taz are activated in Schwann cells by mechanical stimuli and regulate Schwann cell proliferation and transcription of basal lamina receptor genes, both necessary for radial sorting of axons and subsequent myelination. These data link transcriptional effectors of the Hippo pathway and of mechanotransduction to myelin formation in Schwann cells.

Multiple roles for the hippo effector yap in gut regeneration and cancer initiation.

The Hippo signaling effector Yes-associated protein (Yap) is known for its potent control of tissue growth. Our recent work now shows that Yap promotes regeneration in the intestine by reprogramming intestinal stem cells and blocking their terminal differentiation. Similarly, in tumor-initiating cells Yap regenerative signaling synergizes with Wnt activation to drive adenoma formation.

Seeing is believing: Wnt3 localization in the gut epithelium.

Wnt morphogens are notoriously elusive proteins. Thanks to a recent study published in Nature, Clevers and colleagues give us a first glimpse of a mammalian Wnt in action in the gut epithelium.