Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also ...directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1−/− mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1−/− progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1−/− neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1−/−Ripk3−/−, Ripk1−/−Mlkl−/−, and Ripk1−/−Myd88−/− mice prevented neonatal lethality, but only Ripk1−/−Ripk3−/−Casp8−/− mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.
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•RIPK1 delivers a TNF-dependent survival signal to HSC entering the bone marrow•RIPK1 can inhibit RIPK3/MLKL necroptosis•RIPK3/MLKL-dependent necroptosis causes IL-33 release in Ripk1−/− mice•Lethal, necroptosis-induced, systemic inflammation in Ripk1−/− mice is Myd88 dependent
Ripk1 deficiency induces RIPK3/MLKL-dependent necroptosis, triggers IL-33 release, and causes Myd88-dependent systemic inflammation and perinatal death. These phenotypes could be rescued by deletion of Ripk3 and Casp8, revealing a key function for RIPK1 in inhibiting necroptosis and limiting inflammation.
Intra-tumoural heterogeneity and cancer cell plasticity in colorectal cancer (CRC) have been key challenges to effective treatment for patients. It has been suggested that a subpopulation of ...LGR5-expressing cancer stem cells (CSCs) is responsible for driving tumour relapse and therapy resistance in CRC. However, studies have revealed that the LGR5
+ve
CSC population is highly sensitive to chemotherapy. It has been hypothesised that another subset of tumour cells can phenotypically revert to a stem-like state in response to chemotherapy treatment which replenishes the LGR5
+ve
CSC population and maintains tumour growth. Recently, a unique stem cell population marked by enriched clusterin (CLU) expression and termed the revival stem cell (RevSC) was identified in the regenerating murine intestine. This CLU-expressing cell population is quiescent during homeostasis but has the ability to survive and regenerate other stem cells upon injury. More recently, the CLU
+ve
signature has been implicated in several adverse outcomes in CRC, including chemotherapy resistance and poor patient survival; however, the mechanism behind this remains undetermined. In this review, we discuss recent insights on CLU in CRC and its roles in enhancing the plasticity of cells and further consider the implications of CLU as a prospective target for therapeutic intervention.
Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with ...activating mutations in the mitogen-activated kinase pathway gene,
, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein.
We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair.
Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not
alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting.
We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.
Abstract
INPP4B suppresses PI3K/AKT signaling by converting PI(3,4)P
2
to PI(3)P and INPP4B inactivation is common in triple-negative breast cancer. Paradoxically, INPP4B is also a reported oncogene ...in other cancers. How these opposing INPP4B roles relate to PI3K regulation is unclear. We report
PIK3CA
-mutant ER
+
breast cancers exhibit increased INPP4B mRNA and protein expression and INPP4B increased the proliferation and tumor growth of
PIK3CA
-mutant ER
+
breast cancer cells, despite suppression of AKT signaling. We used integrated proteomics, transcriptomics and imaging to demonstrate INPP4B localized to late endosomes via interaction with Rab7, which increased endosomal PI3Kα-dependent PI(3,4)P
2
to PI(3)P conversion, late endosome/lysosome number and cargo trafficking, resulting in enhanced GSK3β lysosomal degradation and activation of Wnt/β-catenin signaling. Mechanistically, Wnt inhibition or depletion of the PI(3)P-effector, Hrs, reduced INPP4B-mediated cell proliferation and tumor growth. Therefore, INPP4B facilitates PI3Kα crosstalk with Wnt signaling in ER
+
breast cancer via PI(3,4)P
2
to PI(3)P conversion on late endosomes, suggesting these tumors may be targeted with combined PI3K and Wnt/β-catenin therapies.
Epidermal Growth Factor (EGF) has long been known for its role in promoting proliferation of intestinal epithelial cells. EGF is produced by epithelial niche cells at the base of crypts
in vivo
and ...is routinely added to the culture medium to support the growth of intestinal organoids
ex vivo
. The recent identification of diverse stromal cell populations that reside underneath intestinal crypts has enabled the characterization of key growth factor cues supplied by these cells. The nature of these signals and how they are delivered to drive intestinal epithelial development, daily homeostasis and tissue regeneration following injury are being investigated. It is clear that aside from EGF, other ligands of the family, including Neuregulin 1 (NRG1), have distinct roles in supporting the function of intestinal stem cells through the ErbB pathway.
Epidermal growth factor (EGF) maintains intestinal stem cell (ISC) proliferation and is a key component of organoid growth media yet is dispensable for intestinal homeostasis, suggesting roles for ...multiple EGF family ligands in ISC function. Here, we identified neuregulin 1 (NRG1) as a key EGF family ligand that drives tissue repair following injury. NRG1, but not EGF, is upregulated upon damage and is expressed in mesenchymal stromal cells, macrophages, and Paneth cells. NRG1 deletion reduces proliferation in intestinal crypts and compromises regeneration capacity. NRG1 robustly stimulates proliferation in crypts and induces budding in organoids, in part through elevated and sustained activation of mitogen-activated protein kinase (MAPK) and AKT. Consistently, NRG1 treatment induces a proliferative gene signature and promotes organoid formation from progenitor cells and enhances regeneration following injury. These data suggest mesenchymal-derived NRG1 is a potent mediator of tissue regeneration and may inform the development of therapies for enhancing intestinal repair after injury.
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•NRG1 is upregulated in cells in the mesenchyme to support efficient tissue regeneration•NRG1 promotes stem cell characteristics in progenitor cells•NRG1 is more potent than EGF in driving cell proliferation in organoids•Exogenous NRG1 enhances regeneration of crypts following injury
Jardé, Abud, and colleagues show that neuregulin 1 (NRG1) is upregulated within distinct mesenchymal niche cells to support intestinal tissue regeneration following damage. NRG1 robustly drives the proliferative response in intestinal stem cells, promotes stem cell identity, and can further enhance the regenerative response when added exogenously.
Gastrointestinal infections often induce epithelial damage that must be repaired for optimal gut function. While intestinal stem cells are critical for this regeneration process R. C. van der Wath, ...B. S. Gardiner, A. W. Burgess, D. W. Smith, PLoS One 8, e73204 (2013); S. Kozar et al., Cell Stem Cell 13, 626–633 (2013), how they are impacted by enteric infections remains poorly defined. Here, we investigate infection-mediated damage to the colonic stem cell compartment and how this affects epithelial repair and recovery from infection. Using the pathogen Clostridioides difficile, we show that infection disrupts murine intestinal cellular organization and integrity deep into the epithelium, to expose the otherwise protected stem cell compartment, in a TcdB-mediated process. Exposure and susceptibility of colonic stem cells to intoxication compromises their function during infection, which diminishes their ability to repair the injured epithelium, shown by altered stem cell signaling and a reduction in the growth of colonic organoids from stem cells isolated from infected mice. We also show, using both mouse and human colonic organoids, that TcdB from epidemic ribotype 027 strains does not require Frizzled 1/2/7 binding to elicit this dysfunctional stem cell state. This stem cell dysfunction induces a significant delay in recovery and repair of the intestinal epithelium of up to 2 wk post the infection peak. Our results uncover a mechanism by which an enteric pathogen subverts repair processes by targeting stem cells during infection and preventing epithelial regeneration, which prolongs epithelial barrier impairment and creates an environment in which disease recurrence is likely.
Several studies have suggested ERBB3/HER3 may be a useful prognostic marker for colorectal cancer. Tumours with an intestinal stem cell signature have also been shown to be more aggressive. Here, we ...investigate whether ERBB3 is associated with intestinal stem cell markers in colorectal cancer and if cancer stem cells within tumours are marked by expression of ERBB3. Expression of ERBB3 and intestinal stem cell markers (LGR5, EPHB2, CD44s and CD44v6) was assessed by qRT-PCR in primary colorectal tumours (stages 0 to IV) and matched normal tissues from 53 patients. The localisation of ERBB3, EPHB2 and KI-67 within tumours was investigated using co-immunofluorescence. Expression of ERBB3 and intestinal stem cell markers were significantly elevated in adenomas and colorectal tumours compared to normal tissue. Positive correlations were found between ERBB3 and intestinal stem cell markers. However, co-immunofluorescence analysis showed that ERBB3 and EPHB2 marked specific cell populations that were mutually exclusive within tumours with distinct proliferative potentials, the majority of ERBB3+ve cells being non-proliferative. This pattern resembles cellular organisation within normal colonic epithelium where EPHB2 labelled proliferative cells reside at the crypt base and ERBB3+ve cells mark differentiated cells at the top of crypts. Our results show that ERBB3 and intestinal stem cell markers correlate in colorectal cancers. ERBB3 localises to differentiated cell populations within tumours that are non-proliferative and distinct from cancer stem cells. These data support the concept that tumours contain discrete stem, proliferative and differentiation compartments similar to that present in normal crypts.
The transmembrane metalloprotease ADAM10 sheds a range of cell surface proteins, including ligands and receptors of the Notch, Eph, and erbB families, thereby activating signaling pathways critical ...for tumor initiation and maintenance. ADAM10 is thus a promising therapeutic target. Although widely expressed, its activity is normally tightly regulated. We now report prevalence of an active form of ADAM10 in tumors compared with normal tissues, in mouse models and humans, identified by our conformation-specific antibody mAb 8C7. Structure/function experiments indicate mAb 8C7 binds an active conformation dependent on disulfide isomerization and oxidative conditions, common in tumors. Moreover, this active ADAM10 form marks cancer stem-like cells with active Notch signaling, known to mediate chemoresistance. Importantly, specific targeting of active ADAM10 with 8C7 inhibits Notch activity and tumor growth in mouse models, particularly regrowth after chemotherapy. Our results indicate targeted inhibition of active ADAM10 as a potential therapy for ADAM10-dependent tumor development and drug resistance.
Organoids are three‐dimensional self‐renewing and organizing clusters of cells that recapitulate the behavior and functionality of developed organs. Referred to as “organs in a dish,” organoids are ...invaluable biological models for disease modeling or drug screening. Currently, organoid culture commonly relies on an expensive and undefined tumor‐derived reconstituted basal membrane which hinders its application in high‐throughput screening, regenerative medicine, and diagnostics. Here, we introduce a novel engineered plant‐based nanocellulose hydrogel is introduced as a well‐defined and low‐cost matrix that supports organoid growth. Gels containing 0.1% nanocellulose fibers (99.9% water) are ionically crosslinked and present mechanical properties similar to the standard animal‐based matrix. The regulation of the osmotic pressure is performed by a salt‐free strategy, offering conditions for cell survival and proliferation. Cellulose nanofibers are functionalized with fibronectin‐derived adhesive sites to provide the required microenvironment for small intestinal organoid growth and budding. Comparative transcriptomic profiling reveals a good correlation with transcriptome‐wide gene expression pattern between organoids cultured in both materials, while differences are observed in stem cells‐specific marker genes. These hydrogels are tunable and can be combined with laminin‐1 and supplemented with insulin‐like growth factor (IGF‐1) to optimize the culture conditions. Nanocellulose hydrogel emerges as a promising matrix for the growth of organoids.
Plant‐based nanocellulose hydrogel is introduced as a well‐defined and very low‐cost porous nanofibrous matrix that supports organoid growth. The mechanical, chemical, and biological properties of the gel are engineered to mimic the extracellular matrix (ECM), providing the required microenvironment for small intestinal organoid culture. This performant hydrogel is tunable with ECM‐derived components, emerging as a promising biomaterial for organoid systems.
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