Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-thrombospondin-1 axis.

Lung stem cells are instructed to produce lineage-specific progeny through unknown factors in their microenvironment. We used clonal 3D cocultures of endothelial cells and distal lung stem cells, bronchioalveolar stem cells (BASCs), to probe the instructive mechanisms. Single BASCs had bronchiolar and alveolar differentiation potential in lung endothelial cell cocultures. Gain- and loss-of-function experiments showed that BMP4-Bmpr1a signaling triggers calcineurin/NFATc1-dependent expression of thrombospondin-1 (Tsp1) in lung endothelial cells to drive alveolar lineage-specific BASC differentiation. Tsp1 null mice exhibited defective alveolar injury repair, confirming a crucial role for the BMP4-NFATc1-TSP1 axis in lung epithelial differentiation and regeneration in vivo. Discovery of this pathway points to methods to direct the derivation of specific lung epithelial lineages from multipotent cells. These findings elucidate a pathway that may be a critical target in lung diseases and provide tools to understand the mechanisms of respiratory diseases at the single-cell level.

Recurrent RhoGAP gene fusion CLDN18-ARHGAP26 promotes RHOA activation and focal adhesion kinase and YAP-TEAD signalling in diffuse gastric cancer.

OBJECTIVE: Genomic studies of gastric cancer have identified highly recurrent genomic alterations impacting RHO signalling, especially in the diffuse gastric cancer (DGC) histological subtype. Among these alterations are interchromosomal translations leading to the fusion of the adhesion protein CLDN18 and RHO regulator ARHGAP26. It remains unclear how these fusion constructs impact the activity of the RHO pathway and what is their broader impact on gastric cancer development. Herein, we developed a model to allow us to study the function of this fusion protein in the pathogenesis of DGC and to identify potential therapeutic targets for DGC tumours with these alterations. DESIGN: We built a transgenic mouse model with LSL-CLDN18-ARHGAP26 fusion engineered into the Col1A1 locus where its expression can be induced by Cre recombinase. Using organoids generated from this model, we evaluated its oncogenic activity and the biochemical effects of the fusion protein on the RHOA pathway and its downstream cell biological effects in the pathogenesis of DGC. RESULTS: We demonstrated that induction of CLDN18-ARHGAP26 expression in gastric organoids induced the formation of signet ring cells, characteristic features of DGC and was able to cooperatively transform gastric cells when combined with the loss of the tumour suppressor geneTrp53. CLDN18-ARHGAP26 promotes the activation of RHOA and downstream effector signalling. Molecularly, the fusion promotes activation of the focal adhesion kinase (FAK) and induction of the YAP pathway. A combination of FAK and YAP/TEAD inhibition can significantly block tumour growth. CONCLUSION: These results indicate that the CLDN18-ARHGAP26 fusion is a gain-of-function DGC oncogene that leads to activation of RHOA and activation of FAK and YAP signalling. These results argue for further evaluation of emerging FAK and YAP-TEAD inhibitors for these deadly cancers.

Down syndrome and leukemia: from basic mechanisms to clinical advances.

Children with Down syndrome (DS, trisomy 21) are at a significantly higher risk of developing acute leukemia compared to the overall population. Many studies investigating the link between trisomy 21 and leukemia initiation and progression have been conducted over the last two decades. Despite improved treatment regimens and significant progress in iden - tifying genes on chromosome 21 and the mechanisms by which they drive leukemogenesis, there is still much that is unknown. A focused group of scientists and clinicians with expertise in leukemia and DS met in October 2022 at the Jérôme Lejeune Foundation in Paris, France for the 1st International Symposium on Down Syndrome and Leukemia. This meeting was held to discuss the most recent advances in treatment regimens and the biology underlying the initiation, progression, and relapse of acute lymphoblastic leukemia and acute myeloid leukemia in children with DS. This review provides a summary of what is known in the field, challenges in the management of DS patients with leukemia, and key questions in the field.

KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis.

Our previous work showed that KRAS activation in gastric cancer cells leads to activation of an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Here we analyze how this KRAS activation in gastric CSCs promotes tumor angiogenesis and metastasis. Gastric cancer CSCs were found to secrete pro-angiogenic factors such as vascular endothelial growth factor A (VEGF-A), and inhibition of KRAS markedly reduced secretion of these factors. In a genetically engineered mouse model, gastric tumorigenesis was markedly attenuated when both KRAS and VEGF-A signaling were blocked. In orthotropic implant and experimental metastasis models, silencing of KRAS and VEGF-A using shRNA in gastric CSCs abrogated primary tumor formation, lymph node metastasis, and lung metastasis far greater than individual silencing of KRAS or VEGF-A. Analysis of gastric cancer patient samples using RNA sequencing revealed a clear association between high expression of the gastric CSC marker CD44 and expression of both KRAS and VEGF-A, and high CD44 and VEGF-A expression predicted worse overall survival. In conclusion, KRAS activation in gastric CSCs enhances secretion of pro-angiogenic factors and promotes tumor progression and metastasis.

Loss of Down syndrome critical region-1 leads to cholesterol metabolic dysfunction that exaggerates hypercholesterolemia in ApoE-null background.

Down syndrome critical region (DSCR)-1 functions as a feedback modulator for calcineurin-nuclear factor for activated T cell (NFAT) signals, which are crucial for cell proliferation and inflammation. Stable expression of DSCR-1 inhibits pathological angiogenesis and septic inflammation. DSCR-1 also plays a critical role in vascular wall remodeling associated with aneurysm development that occurs primarily in smooth muscle cells. Besides, Dscr-1 deficiency promotes the M1-to M2-like phenotypic switch in macrophages, which correlates to the reduction of denatured cholesterol uptakes. However, the distinct roles of DSCR-1 in cholesterol and lipid metabolism are not well understood. Here, we show that loss of apolipoprotein (Apo) E in mice with chronic hypercholesterolemia induced Dscr-1 expression in the liver and aortic atheroma. In Dscr-1-null mice fed a high-fat diet, oxidative- and endoplasmic reticulum (ER) stress was induced, and sterol regulatory element-binding protein (SREBP) 2 production in hepatocytes was stimulated. This exaggerated ApoE-/--mediated nonalcoholic fatty liver disease (NAFLD) and subsequent hypercholesterolemia. Genome-wide screening revealed that loss of both ApoE and Dscr-1 resulted in the induction of immune- and leukocyte activation-related genes in the liver compared with ApoE deficiency alone. However, expressions of inflammation-activated markers and levels of monocyte adhesion were suspended upon induction of the Dscr-1 null background in the aortic endothelium. Collectively, our study shows that the combined loss of Dscr-1 and ApoE causes metabolic dysfunction in the liver but reduces atherosclerotic plaques, thereby leading to a dramatic increase in serum cholesterol and the formation of sporadic vasculopathy.

Short and long-term outcomes of prophylactic total gastrectomy in 54 consecutive individuals with germline pathogenic mutations in the CDH1 gene.

BACKGROUND: Germline mutation of CDH1 is rare and leads to hereditary diffuse gastric cancer (DGC). METHODS: Patients (pts) with CDH1 mutation who underwent multidisciplinary counseling followed by open prophylactic total gastrectomy (PTG) by a single surgeon were reviewed. RESULTS: Fifty-four pts with a median age of 41 years (16-70 years) underwent PTG between 2006 and 2021. Median operative time was 161 min, and median hospital stay was 7 days (range 6-12). There were 5 complications (9.2%) within 30 days, and two complications (pulmonary embolism and pancreatitis) required readmission. There were no anastomotic leaks. The pathologic analysis of the first 10 pts included the entire gastric mucosa, revealing a median of 15 foci of DGC (range 5-136). The subsequent 44 pts with more limited analysis had a median of 2 foci (range 0-5), and two pts (3.7%) had no foci identified. Median maximum weight loss was 19%. In long-term follow-up (median 4.6 years) of 20 pts, median global QOL was 2.0 (very good), the majority had persistent difficulty with certain foods or liquids, and all stated they would again elect PTG over surveillance endoscopy. CONCLUSIONS: PTG can be performed safely at high-volume referral centers with very good QOL but nutritional sequelae persist.

WITHDRAWN: FOXC1 modulates stem-like cell properties and chemoresistance through hedgehog and EMT signaling in gastric adenocarcinoma.

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Loss of Down Syndrome Critical Region-1 Mediated-Hypercholesterolemia Accelerates Corneal Opacity Via Pathological Neovessel Formation.

OBJECTIVE: The calcineurin-NFAT (nuclear factor for activated T cells)-DSCR (Down syndrome critical region)-1 pathway plays a crucial role as the downstream effector of VEGF (vascular endothelial growth factor)-mediated tumor angiogenesis in endothelial cells. A role for DSCR-1 in different organ microenvironment such as the cornea and its role in ocular diseases is not well understood. Corneal changes can be indicators of various disease states and are easily detected through ocular examinations. Approach and Results: The presentation of a corneal arcus or a corneal opacity due to lipid deposition in the cornea often indicates hyperlipidemia and in most cases, hypercholesterolemia. Although the loss of Apo (apolipoprotein) E has been well characterized and is known to lead to elevated serum cholesterol levels, there are few corneal changes observed in ApoE-/- mice. In this study, we show that the combined loss of ApoE and DSCR-1 leads to a dramatic increase in serum cholesterol levels and severe corneal opacity with complete penetrance. The cornea is normally maintained in an avascular state; however, loss of Dscr-1 is sufficient to induce hyper-inflammatory and -oxidative condition, increased corneal neovascularization, and lymphangiogenesis. Furthermore, immunohistological analysis and genome-wide screening revealed that loss of Dscr-1 in mice triggers increased immune cell infiltration and upregulation of SDF (stromal derived factor)-1 and its receptor, CXCR4 (C-X-C motif chemokine ligand receptor-4), potentiating this signaling axis in the cornea, thereby contributing to pathological corneal angiogenesis and opacity. CONCLUSIONS: This study is the first demonstration of the critical role for the endogenous inhibitor of calcineurin, DSCR-1, and pathological corneal angiogenesis in hypercholesterolemia induced corneal opacity.

CDK5RAP3 as tumour suppressor negatively regulates self-renewal and invasion and is regulated by ERK1/2 signalling in human gastric cancer.

BACKGROUND: Toward identifying new strategies to target gastric cancer stem-like cells (CSCs), we evaluated the function of the tumour suppressor CDK5 regulatory subunit-associated protein 3 (CDK5RAP3) in gastric CSC maintenance. METHODS: We examined the expression of CDK5RAP3 and CD44 in gastric cancer patients. The function and mechanisms of CDK5RAP3 were checked in human and mouse gastric cancer cell lines and in mouse xenograft. RESULTS: We show that CDK5RAP3 is weakly expressed in gastric CSCs and is negatively correlated with the gastric CSC marker CD44. CDK5RAP3 overexpression decreased expression of CSC markers, spheroid formation, invasion and migration, and reversed chemoresistance in gastric CSCs in vitro and vivo. CDK5RAP3 expression was found to be regulated by extracellular-related kinase (ERK) signalling. ERK inhibitors decreased spheroid formation, migration and invasion, and the expression of epithelial-to-mesenchymal transition (EMT)-related proteins in both GA cells and organoids derived from a genetically engineered mouse model of GA. Finally, CDK5RAP3 expression was associated with reduced lymph-node metastasis and better prognosis, even in the presence of high expression of the EMT transcription factor Snail, among patients with CD44-positive GA. CONCLUSIONS: Our results demonstrate that CDK5RAP3 is suppressed by ERK signalling and negatively regulates the self-renewal and EMT of gastric CSCs.

Testicular endothelial cells promote self-renewal of spermatogonial stem cells in rats†.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis in male due to their capability to multiply in numbers by self-renewal and subsequent meiotic processes. However, as SSCs are present in a very small proportion in the testis, in vitro proliferation of undifferentiated SSCs will facilitate the study of germ cell biology. In this study, we investigated the effectiveness of various cell lines as a feeder layer for rat SSCs. Germ cells enriched for SSCs were cultured on feeder layers including SIM mouse embryo-derived thioguanine and ouabain-resistant cells, C166 cells, and mouse and rat testicular endothelial cells (TECs) and their stem cell potential for generating donor-derived colonies and offspring was assessed by transplantation into recipient testes. Rat germ cells cultured on TECs showed increased mRNA and protein levels of undifferentiated spermatogonial markers. Rat SSCs derived from these germ cells underwent spermatogenesis and generated offspring when transplanted into recipients. Collectively, TECs can serve as an effective feeder layer that enhances the proliferative and self-renewal capacity of cultured rat SSCs while preserving their stemness properties.

Translational Upregulation of an Individual p21Cip1 Transcript Variant by GCN2 Regulates Cell Proliferation and Survival under Nutrient Stress.

Multiple transcripts encode for the cell cycle inhibitor p21(Cip1). These transcripts produce identical proteins but differ in their 5' untranslated regions (UTRs). Although several stresses that induce p21 have been characterized, the mechanisms regulating the individual transcript variants and their functional significance are unknown. Here we demonstrate through (35)S labeling, luciferase reporter assays, and polysome transcript profiling that activation of the Integrated Stress Response (ISR) kinase GCN2 selectively upregulates the translation of a p21 transcript variant containing 5' upstream open reading frames (uORFs) through phosphorylation of the eukaryotic translation initiation factor eIF2α. Mutational analysis reveals that the uORFs suppress translation under basal conditions, but promote translation under stress. Functionally, ablation of p21 ameliorates G1/S arrest and reduces cell survival in response to GCN2 activation. These findings uncover a novel mechanism of p21 post-transcriptional regulation, offer functional significance for the existence of multiple p21 transcripts, and support a key role for GCN2 in regulating the cell cycle under stress.

The effect of environmental chemicals on the tumor microenvironment.

Potentially carcinogenic compounds may cause cancer through direct DNA damage or through indirect cellular or physiological effects. To study possible carcinogens, the fields of endocrinology, genetics, epigenetics, medicine, environmental health, toxicology, pharmacology and oncology must be considered. Disruptive chemicals may also contribute to multiple stages of tumor development through effects on the tumor microenvironment. In turn, the tumor microenvironment consists of a complex interaction among blood vessels that feed the tumor, the extracellular matrix that provides structural and biochemical support, signaling molecules that send messages and soluble factors such as cytokines. The tumor microenvironment also consists of many host cellular effectors including multipotent stromal cells/mesenchymal stem cells, fibroblasts, endothelial cell precursors, antigen-presenting cells, lymphocytes and innate immune cells. Carcinogens can influence the tumor microenvironment through effects on epithelial cells, the most common origin of cancer, as well as on stromal cells, extracellular matrix components and immune cells. Here, we review how environmental exposures can perturb the tumor microenvironment. We suggest a role for disrupting chemicals such as nickel chloride, Bisphenol A, butyltins, methylmercury and paraquat as well as more traditional carcinogens, such as radiation, and pharmaceuticals, such as diabetes medications, in the disruption of the tumor microenvironment. Further studies interrogating the role of chemicals and their mixtures in dose-dependent effects on the tumor microenvironment could have important general mechanistic implications for the etiology and prevention of tumorigenesis.

The calcineurin-NFAT pathway negatively regulates megakaryopoiesis.

The calcium regulated calcineurin-nuclear factor of activated T cells (NFAT) pathway modulates the physiology of numerous cell types, including hematopoietic. Upon activation, calcineurin dephosphorylates NFAT family transcription factors, triggering their nuclear entry and activation or repression of target genes. NFATc1 and c2 isoforms are expressed in megakaryocytes. Moreover, human chromosome 21 (Hsa21) encodes several negative regulators of calcineurin-NFAT, candidates in the pathogenesis of Down syndrome (trisomy 21)-associated transient myeloproliferative disorder and acute megakaryoblastic leukemia. To investigate the role of calcineurin-NFAT in megakaryopoiesis, we examined wild-type mice treated with the calcineurin inhibitor cyclosporin A and transgenic mice expressing a targeted single extra copy of Dscr1, an Hsa21-encoded calcineurin inhibitor. Both murine models exhibited thrombocytosis with increased megakaryocytes and megakaryocyte progenitors. Pharmacological or genetic inhibition of calcineurin in mice caused accumulation of megakaryocytes exhibiting enhanced 5-bromo-2'-deoxyuridine uptake and increased expression of messenger RNAs encoding CDK4 and G1 cyclins, which promote cell division. Additionally, human megakaryocytes with trisomy 21 show increased proliferation and decreased NFAT activation compared with euploid controls. Our data indicate that inhibition of calcineurin-NFAT drives proliferation of megakaryocyte precursors by de-repressing genes that drive cell division, providing insights into mechanisms of normal megakaryopoiesis and megakaryocytic abnormalities that accompany Down syndrome.

Down's syndrome suppression of tumour growth and the role of the calcineurin inhibitor DSCR1.

The incidence of many cancer types is significantly reduced in individuals with Down's syndrome, and it is thought that this broad cancer protection is conferred by the increased expression of one or more of the 231 supernumerary genes on the extra copy of chromosome 21. One such gene is Down's syndrome candidate region-1 (DSCR1, also known as RCAN1), which encodes a protein that suppresses vascular endothelial growth factor (VEGF)-mediated angiogenic signalling by the calcineurin pathway. Here we show that DSCR1 is increased in Down's syndrome tissues and in a mouse model of Down's syndrome. Furthermore, we show that the modest increase in expression afforded by a single extra transgenic copy of Dscr1 is sufficient to confer significant suppression of tumour growth in mice, and that such resistance is a consequence of a deficit in tumour angiogenesis arising from suppression of the calcineurin pathway. We also provide evidence that attenuation of calcineurin activity by DSCR1, together with another chromosome 21 gene Dyrk1a, may be sufficient to markedly diminish angiogenesis. These data provide a mechanism for the reduced cancer incidence in Down's syndrome and identify the calcineurin signalling pathway, and its regulators DSCR1 and DYRK1A, as potential therapeutic targets in cancers arising in all individuals.

Concomitant low-dose doxorubicin treatment and exercise.

Cardiotoxicity is a side effect for cancer patients treated with doxorubicin (DOX). We tested the hypothesis that low-intensity aerobic exercise concomitant with DOX treatment would offset DOX-induced cardiotoxicity while also improving the therapeutic efficacy of DOX on tumor progression. B16F10 melanoma cells (3 × 10(5)) were injected subcutaneously into the scruff of 6- to 8-wk-old male C57BL/6 mice (n = 48). A 4 mg/kg cumulative dose of DOX was administered over 2 wk, and exercise (EX) consisted of treadmill walking (10 m/min, 45 min/day, 5 days/wk, 2 wk). Four experimental groups were tested: 1) sedentary (SED) + vehicle, 2) SED + DOX, 3) EX + vehicle, and 4) EX + DOX. Tumor volume was attenuated in DOX and lowest in EX + DOX. DOX-treated animals had less gain in body weight, reduced heart weights (HW), smaller HW-to-body weight ratios, and shorter tibial lengths by the end of the protocol; and exercise did not reverse the cardiotoxic effects of DOX. Despite decreased left ventricular (LV) mass with DOX, cardiomyocyte cross-sectional area, ß-myosin heavy chain gene expression, and whole heart systolic (fractional shortening) and diastolic (E/A ratio) function were similar among groups. DOX also resulted in increased LV fibrosis with lower LV end diastolic volume and stroke volume. Myocardial protein kinase B activity was increased with both DOX and EX treatments, and tuberous sclerosis 2 (TSC2) abundance was reduced with EX. Downstream phosphorylation of TSC2 and mammalian target of rapamycin were similar across groups. We conclude that exercise increases the efficacy of DOX in inhibiting tumor growth without mitigating subclinical DOX-induced cardiotoxicity in a murine model of melanoma.

Establishment and Characterization of Patient-derived Gastric Organoids from Biopsies of Benign Gastric Body and Antral Epithelium.

Gastric patient-derived organoids (PDOs) offer a unique tool for studying gastric biology and pathology. Consequently, these PDOs find increasing use in a wide array of research applications. However, a shortage of published approaches exists for producing gastric PDOs from single-cell digests while maintaining a standardized initial cell seeding density. In this protocol, the emphasis is on the initiation of gastric organoids from isolated single cells and the provision of a method for passaging organoids through fragmentation. Importantly, the protocol demonstrates that a standardized approach to the initial cell seeding density consistently yields gastric organoids from benign biopsy tissue and allows for standardized quantification of organoid growth. Finally, evidence supports the novel observation that gastric PDOs display varying rates of formation and growth based on whether the organoids originate from biopsies of the body or antral regions of the stomach. Specifically, it is revealed that the use of antral biopsy tissue for organoid initiation results in a greater number of organoids formed and more rapid organoid growth over a 20-day period when compared to organoids generated from biopsies of the gastric body. The protocol described herein offers investigators a timely and reproducible method for successfully generating and working with gastric PDOs.

Diffuse Gastric Cancer: A Comprehensive Review of Molecular Features and Emerging Therapeutics.

Diffuse-type gastric cancer (DGC) accounts for approximately one-third of gastric cancer diagnoses but is a more clinically aggressive disease with peritoneal metastases and inferior survival compared with intestinal-type gastric cancer (IGC). The understanding of the pathogenesis of DGC has been relatively limited until recently. Multiomic studies, particularly by The Cancer Genome Atlas, have better characterized gastric adenocarcinoma into molecular subtypes. DGC has unique molecular features, including alterations in CDH1, RHOA, and CLDN18-ARHGAP26 fusions. Preclinical models of DGC characterized by these molecular alterations have generated insight into mechanisms of pathogenesis and signaling pathway abnormalities. The currently approved therapies for treatment of gastric cancer generally provide less clinical benefit in patients with DGC. Based on recent phase II/III clinical trials, there is excitement surrounding Claudin 18.2-based and FGFR2b-directed therapies, which capitalize on unique biomarkers that are enriched in the DGC populations. There are numerous therapies targeting Claudin 18.2 and FGFR2b in various stages of preclinical and clinical development. Additionally, there have been preclinical advancements in exploiting unique therapeutic vulnerabilities in several models of DGC through targeting of the focal adhesion kinase (FAK) and Hippo pathways. These preclinical and clinical advancements represent a promising future for the treatment of DGC.

Nociceptive neurons interact directly with gastric cancer cells via a CGRP/Ramp1 axis to promote tumor progression.

Cancer cells have been shown to exploit neurons to modulate their survival and growth, including through establishment of neural circuits within the central nervous system (CNS) 1-3 . Here, we report a distinct pattern of cancer-nerve interactions between the peripheral nervous system (PNS) and gastric cancer (GC). In multiple GC mouse models, nociceptive nerves demonstrated the greatest degree of nerve expansion in an NGF-dependent manner. Neural tracing identified CGRP+ peptidergic neurons as the primary gastric sensory neurons. Three-dimensional co-culture models showed that sensory neurons directly connect with gastric cancer spheroids through synapse-like structures. Chemogenetic activation of sensory neurons induced the release of calcium into the cytoplasm of cancer cells, promoting tumor growth and metastasis. Pharmacological ablation of sensory neurons or treatment with CGRP inhibitors suppressed tumor growth and extended survival. Depolarization of gastric tumor membranes through in vivo optogenetic activation led to enhanced calcium flux in nodose ganglia and CGRP release, defining a cancer cell-peptidergic neuronal circuit. Together, these findings establish the functional connectivity between cancer and sensory neurons, identifying this pathway as a potential therapeutic target.