Verteporfin inhibits TGF-ß signaling by disrupting the Smad2/3-Smad4 interaction.

Transforming growth factor-ß (TGF-ß) signaling plays a crucial role in pathogenesis, such as accelerating tissue fibrosis and promoting tumor development at the later stages of tumorigenesis by promoting epithelial-mesenchymal transition, cancer cell migration, and invasion. Targeting TGF-ß signaling is a promising therapeutic approach, but non-specific inhibition may result in adverse effects. In this study, we focus on the Smad2/3-Smad4 complex, a key component in TGF-ß signaling transduction, as a potential target for cancer therapy. Through a phase-separated condensate-aided biomolecular interaction system, we identified verteporfin (VP) as a small-molecule inhibitor that specifically targets the Smad2/3-Smad4 interaction. VP effectively disrupted the interaction between Smad2/3 and Smad4 and thereby inhibited canonical TGF-ß signaling, but not the interaction between Smad1 and Smad4 in BMP signaling. Furthermore, VP exhibited inhibitory effects on TGF-ß-induced epithelial-mesenchymal transition and cell migration. Our findings indicate a novel approach to develop protein-protein interaction inhibitors of the canonical TGF-ß signaling pathway for treatments of related diseases.

Upregulation of CYR61 by TGF-ß and YAP signaling exerts a counter-suppression of hepatocellular carcinoma.

Transforming growth factor-ß (TGF-ß) and Hippo signaling are two critical pathways engaged in cancer progression by regulating both oncogenes and tumor suppressors, yet how the two pathways coordinately exert their functions in the development of hepatocellular carcinoma (HCC) remains elusive. In this study, we firstly conducted an integrated analysis of public liver cancer databases and our experimental TGF-ß target genes, identifying CYR61 as a pivotal candidate gene relating to HCC development. The expression of CYR61 is downregulated in clinical HCC tissues and cell lines than that in the normal counterparts. Evidence revealed that CYR61 is a direct target gene of TGF-ß in liver cancer cells. In addition, TGF-ß-stimulated Smad2/3 and the Hippo pathway downstream effectors YAP and TEAD4 can form a protein complex on the promoter of CYR61, thereby activating the promoter activity and stimulating CYR61 gene transcription in a collaborative manner. Functionally, depletion of CYR61 enhanced TGF-ß- or YAP-mediated growth and migration of liver cancer cells. Consistently, ectopic expression of CYR61 was capable of impeding TGF-ß- or YAP-induced malignant transformation of HCC cells in vitro and attenuating HCC xenograft growth in nude mice. Finally, transcriptomic analysis indicates that CYR61 can elicit an antitumor program in liver cancer cells. Together, these results add new evidence for the crosstalk between TGF-ß and Hippo signaling and unveil an important tumor suppressor function of CYR61 in liver cancer.

A growth factor-reduced culture system for colorectal cancer organoids.

Although organoids derived from tumor tissues have been widely used in cancer research, it is a great challenge for cultured organoids to retain the characteristics of the original tumor tissues due to their heterogeneity. In this study, we explore organoid culture recipes to capture tumor features of colorectal cancers. We find that the activation of Wnt and EGF signaling and inhibition of BMP signaling are non-essential for the survival of most colorectal cancer organoids (CRCOs). We design a growth factor-reduced culture medium containing FGF10, A83-01 (TGF-ß type I receptor inhibitor), SB202190 (p38 MAPK inhibitor), gastrin, and nicotinamide. Using this medium, we can maintain tumor features in long-term CRCO cultivation, as evidenced by histopathology, genetic stability, tumorigenicity, and response of clinical treatments. Our findings offer a reliable and economical strategy for CRCO culture, facilitating the utilization of organoids in colorectal cancer research and treatment.

HER2 phosphorylation induced by TGF-ß promotes mammary morphogenesis and breast cancer progression.

Transforming growth factor ß (TGF-ß) and HER2 signaling collaborate to promote breast cancer progression. However, their molecular interplay is largely unclear. TGF-ß can activate mitogen-activated protein kinase (MAPK) and AKT, but the underlying mechanism is not fully understood. In this study, we report that TGF-ß enhances HER2 activation, leading to the activation of MAPK and AKT. This process depends on the TGF-ß type I receptor TßRI kinase activity. TßRI phosphorylates HER2 at Ser779, promoting Y1248 phosphorylation and HER2 activation. Mice with HER2 S779A mutation display impaired mammary morphogenesis, reduced ductal elongation, and branching. Furthermore, wild-type HER2, but not S779A mutant, promotes TGF-ß-induced epithelial-mesenchymal transition, cell migration, and lung metastasis of breast cells. Increased HER2 S779 phosphorylation is observed in human breast cancers and positively correlated with the activation of HER2, MAPK, and AKT. Our findings demonstrate the crucial role of TGF-ß-induced S779 phosphorylation in HER2 activation, mammary gland development, and the pro-oncogenic function of TGF-ß in breast cancer progression.

Intraepithelial lymphocytes promote intestinal regeneration through CD160/HVEM signaling.

Chemotherapy and radiotherapy frequently lead to intestinal damage. The mechanisms governing the repair or regeneration of intestinal damage are still not fully elucidated. Intraepithelial lymphocytes (IELs) are the primary immune cells residing in the intestinal epithelial layer. However, whether IELs are involved in intestinal epithelial injury repair remains unclear. Here, we found that IELs rapidly infiltrated the intestinal crypt region and are crucial for the recovery of the intestinal epithelium post-chemotherapy. Interestingly, IELs predominantly promoted intestinal regeneration by modulating the proliferation of transit-amplifying (TA) cells. Mechanistically, the expression of CD160 on IELs allows for interaction with herpes virus entry mediator (HVEM) on the intestinal epithelium, thereby activating downstream nuclear factor kappa (NF-κB) signaling and further promoting intestinal regeneration. Deficiency in either CD160 or HVEM resulted in reduced proliferation of intestinal progenitor cells, impaired intestinal damage repair, and increased mortality following chemotherapy. Remarkably, the adoptive transfer of CD160-sufficient IELs rescued the Rag1 deficient mice from chemotherapy-induced intestinal inflammation. Overall, our study underscores the critical role of IELs in intestinal regeneration and highlights the potential applications of targeting the CD160-HVEM axis for managing intestinal adverse events post-chemotherapy and radiotherapy.

MicroRNA-200 Loaded Lipid Nanoparticles Promote Intestinal Epithelium Regeneration in Canonical MicroRNA-Deficient Mice.

Intestinal epithelium undergoes regeneration after injuries, and the disruption of this process can lead to inflammatory bowel disease and tumorigenesis. Intestinal stem cells (ISCs) residing in the crypts are crucial for maintaining the intestinal epithelium's homeostasis and promoting regeneration upon injury. However, the precise role of DGCR8, a critical component in microRNA (miRNA) biogenesis, in intestinal regeneration remains poorly understood. In this study, we provide compelling evidence demonstrating the indispensable role of epithelial miRNAs in the regeneration of the intestine in mice subjected to 5-FU or irradiation-induced injury. Through a comprehensive pooled screen of miRNA function in Dgcr8-deficient organoids, we observe that the loss of the miR-200 family leads to the hyperactivation of the p53 pathway, thereby reducing ISCs and impairing epithelial regeneration. Notably, downregulation of the miR-200 family and hyperactivation of the p53 pathway are verified in colonic tissues from patients with active ulcerative colitis (UC). Most importantly, the transient supply of miR-200 through the oral delivery of lipid nanoparticles (LNPs) carrying miR-200 restores ISCs and promotes intestinal regeneration in mice following acute injury. Our study implies the miR-200/p53 pathway as a promising therapeutic target for active UC patients with diminished levels of the miR-200 family. Furthermore, our findings suggest that the clinical application of LNP-miRNAs could enhance the efficacy, safety, and acceptability of existing therapeutic modalities for intestinal diseases.

Stromal BMP signaling regulates mucin production in the large intestine via interleukin-1/17.

Bone morphogenic protein (BMP) signaling is critical for intestinal development, homeostasis, and function performance. Although the function of BMP signaling in the intestinal epithelium is well appreciated, the direct effect of BMP on intestinal stromal cells is poorly understood. Here, we show that disruption of BMP signaling by genetic ablation of Alk3 or Smad4 expands the stromal cell pool, the mucosa tumefaction, and colonic polyposis in the large intestine. Interleukin (IL) secretion by stromal cells is notably increased, including IL-1, IL-11, and IL-17. Specifically, IL-1 and IL-17a hyperactivate the mucin production by goblet cells through nuclear factor κB signaling, and abnormal mucin accumulation results in the morphological changes, epithelial barrier destruction, and polyposis development. Together, our results provide an insight into the role of BMP signaling in intestinal stromal cells to regulate epithelium function. This study further highlights the role of mucin-producing goblet cells in intestinal homeostasis and colitis development.

The cyclooxygenase-expressing mesenchyme resists intestinal epithelial injury by paracrine signaling.

Paracrine signals play pivotal roles in organ homeostasis. Mesenchymal stromal cells (MSCs) play a key role in regulating epithelium homeostasis in the intestine while their paracrine effects are poorly characterized. Here, we identified prostaglandin E2 (PGE2) secreted by cyclooxygenase (COX)-expressing MSCs as a vital factor to maintain the intestinal mucosal barrier. We found that MSCs-induced organoid swelling through paracrine effect in vitro, a process due to enhanced water adsorption and is mediated by the COX-PGE2-EP4 axis. To further explore the regulatory effect of this axis on the intestinal epithelial barrier in vivo, we established the conditional knockout mouse model to specifically delete COX in MSCs and found that PGE2 reduction downregulated the gene Muc2 and induced a gastric metaplasia-like phenotype. Moreover, PGE2 defects increased the susceptibility of intestinal epithelium to colitis. Our study uncovers the paracrine signaling of COX-expressing MSCs in intestinal mucosal barrier maintenance, providing a basis for understanding the role of mesenchymal cells in the pathophysiological function of the intestine.

Transforming Growth Factor Beta Promotes Inflammation and Tumorigenesis in Smad4-Deficient Intestinal Epithelium in a YAP-Dependent Manner.

Transforming growth factor beta (TGF-ß), a multifunctional cytokine, plays critical roles in immune responses. However, the precise role of TGF-ß in colitis and colitis-associated cancer remains poorly defined. Here, it is demonstrated that TGF-ß promotes the colonic inflammation and related tumorigenesis in the absence of Smad family member 4 (Smad4). Smad4 loss in intestinal epithelium aggravates colitis and colitis-associated neoplasia induced by dextran sulfate sodium (DSS) and azoxymethane/dextran sulfate sodium (AOM/DSS), leading to over-activated immune responses and increased TGF-ß1 levels. In Smad4-deficient organoids, TGF-ß1 stimulates spheroid formation and impairs intestinal stem cell proliferation and lineage specification. YAP, whose expression is directly upregulated by TGF-ß1 after Smad4 deletion, mediates the effect of TGF-ß1 by interacting with Smad2/3. Attenuation of YAP/TAZ prevents TGF-ß1-induced spheroid formation in Smad4-/- organoids and alleviates colitis and colitis-associated cancer in Smad4-deficient mice. Collectively, these results highlight an integral role of the TGF-ß/Smad4 axis in restraining intestinal inflammation and tumorigenesis and suggest TGF-ß or YAP signaling as therapeutic targets for these gastrointestinal diseases intervention.

Standard: Human intestinal cancer organoids.

Intestinal cancer is one of the most frequent and lethal types of cancer. Modeling intestinal cancer using organoids has emerged in the last decade. Human intestinal cancer organoids are physiologically relevant in vitro models, which provides an unprecedented opportunity for fundamental and applied research in colorectal cancer. "Human intestinal cancer organoids" is the first set of guidelines on human intestinal organoids in China, jointly drafted and agreed by the experts from the Chinese Society for Cell Biology and its branch society: the Chinese Society for Stem Cell Research. This standard specifies terms and definitions, technical requirements, test methods for human intestinal cancer organoids, which apply to the production and quality control during the process of manufacturing and testing of human intestinal cancer organoids. It was released by the Chinese Society for Cell Biology on 24 September 2022. We hope that the publication of this standard will guide institutional establishment, acceptance and execution of proper practocal protocols, and accelerate the international standardization of human intestinal cancer organoids for clinical development and therapeutic applications.

ARL13B promotes angiogenesis and glioma growth by activating VEGFA-VEGFR2 signaling.

BACKGROUND: Tumor angiogenesis is essential for solid tumor progression, invasion and metastasis. The aim of this study was to identify potential signaling pathways involved in tumor angiogenesis. METHODS: Genetically engineered mouse models were used to investigate the effects of endothelial ARL13B(ADP-ribosylation factor-like GTPase 13B) over-expression and deficiency on retinal and cerebral vasculature. An intracranially transplanted glioma model and a subcutaneously implanted melanoma model were employed to examine the effects of ARL13B on tumor growth and angiogenesis. Immunohistochemistry was used to measure ARL13B in glioma tissues, and scRNA-seq was used to analyze glioma and endothelial ARL13B expression. GST-fusion protein-protein interaction and co-immunoprecipitation assays were used to determine the ARL13B-VEGFR2 interaction. Immunobloting, qPCR, dual-luciferase reporter assay and functional experiments were performed to evaluate the effects of ARL13B on VEGFR2 activation. RESULTS: Endothelial ARL13B regulated vascular development of both the retina and brain in mice. Also, ARL13B in endothelial cells regulated the growth of intracranially transplanted glioma cells and subcutaneously implanted melanoma cells by controlling tumor angiogenesis. Interestingly, this effect was attributed to ARL13B interaction with VEGFR2, through which ARL13B regulated the membrane and ciliary localization of VEGFR2 and consequently activated its downstream signaling in endothelial cells. Consistent with its oncogenic role, ARL13B was highly expressed in human gliomas, which was well correlated with the poor prognosis of glioma patients. Remarkably, ARL13B, transcriptionally regulated by ZEB1, enhanced the expression of VEGFA by activating Hedgehog signaling in glioma cells. CONCLUSIONS: ARL13B promotes angiogenesis and tumor growth by activating VEGFA-VEGFR2 signaling. Thus, targeting ARL13B might serve as a potential approach for developing an anti-glioma or anti-melanoma therapy.

Intestinal cellular heterogeneity and disease development revealed by single-cell technology.

The intestinal epithelium is responsible for food digestion and nutrient absorption and plays a critical role in hormone secretion, microorganism defense, and immune response. These functions depend on the integral single-layered intestinal epithelium, which shows diversified cell constitution and rapid self-renewal and presents powerful regeneration plasticity after injury. Derailment of homeostasis of the intestine epithelium leads to the development of diseases, most commonly including enteritis and colorectal cancer. Therefore, it is important to understand the cellular characterization of the intestinal epithelium at the molecular level and the mechanisms underlying its homeostatic maintenance. Single-cell technologies allow us to gain molecular insights at the single-cell level. In this review, we summarize the single-cell RNA sequencing applications to understand intestinal cell characteristics, spatiotemporal evolution, and intestinal disease development.

The Interplay Between TGF-ß Signaling and Cell Metabolism.

The transforming growth factor-ß (TGF-ß) signaling plays a critical role in the development and tissue homeostasis in metazoans, and deregulation of TGF-ß signaling leads to many pathological conditions. Mounting evidence suggests that TGF-ß signaling can actively alter metabolism in diverse cell types. Furthermore, metabolic pathways, beyond simply regarded as biochemical reactions, are closely intertwined with signal transduction. Here, we discuss the role of TGF-ß in glucose, lipid, amino acid, redox and polyamine metabolism with an emphasis on how TGF-ß can act as a metabolic modulator and how metabolic changes can influence TGF-ß signaling. We also describe how interplay between TGF-ß signaling and cell metabolism regulates cellular homeostasis as well as the progression of multiple diseases, including cancer.

Recent advances in tissue stem cells.

Stem cells are undifferentiated cells capable of self-renewal and differentiation, giving rise to specialized functional cells. Stem cells are of pivotal importance for organ and tissue development, homeostasis, and injury and disease repair. Tissue-specific stem cells are a rare population residing in specific tissues and present powerful potential for regeneration when required. They are usually named based on the resident tissue, such as hematopoietic stem cells and germline stem cells. This review discusses the recent advances in stem cells of various tissues, including neural stem cells, muscle stem cells, liver progenitors, pancreatic islet stem/progenitor cells, intestinal stem cells, and prostate stem cells, and the future perspectives for tissue stem cell research.

Targeting hyperactive TGFBR2 for treating MYOCD deficient lung cancer.

Purpose: Clinical success of cancer therapy is severely limited by drug resistance, attributed in large part to the loss of function of tumor suppressor genes (TSGs). Developing effective strategies to treat those tumors is challenging, but urgently needed in clinic. Experimental Design: MYOCD is a clinically relevant TSG in lung cancer patients. Our in vitro and in vivo data confirm its tumor suppressive function. Further analysis reveals that MYOCD potently inhibits stemness of lung cancer stem cells. Mechanistically, MYOCD localizes to TGFBR2 promoter region and thereby recruits PRMT5/MEP50 complex to epigenetically silence its transcription. Conclusions: NSCLC cells deficient of MYOCD are particularly sensitive to TGFBR kinase inhibitor (TGFBRi). TGFBRi and stemness inhibitor synergize with existing drugs to treat MYOCD deficient lung cancers. Our current work shows that loss of function of MYOCD creates Achilles' heels in lung cancer cells, which might be exploited in clinic.

Non-muscle myosin heavy chain 9 maintains intestinal homeostasis by preventing epithelium necroptosis and colitis adenoma formation.

Non-muscle myosin IIA plays an important role in cell adhesion, cell migration, and tissue architecture. We previously showed that low activity of the heavy chain of non-muscle myosin II Myh9 is beneficial to LGR5+ intestinal stem cell maintenance. However, the function of Myh9 in adult mouse intestinal epithelium is largely unclear. In this study, we used the inducible Villin-creERT2 knockout approach to delete Myh9 in adult mouse intestinal epithelium and observed that homozygous deletion of Myh9 causes colitis-like morphologic changes in intestine, leads to a high sensitivity to dextran sulfate sodium and promotes colitis-related adenoma formation in the colon. Myh9 deletion disturbs cell junctions and impairs intestinal lumen barrier integrity, promoting the necroptosis of epithelial cells. Consistently, these changes can be partially rescued by Ripk3 knockout. Our results indicate that Myh9 is required for the maintenance of intestinal epithelium integrity and the prevention of cell necroptosis.

Phase separation of Axin organizes the ß-catenin destruction complex.

In Wnt/ß-catenin signaling, the ß-catenin protein level is deliberately controlled by the assembly of the multiprotein ß-catenin destruction complex composed of Axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3ß (GSK3ß), casein kinase 1α (CK1α), and others. Here we provide compelling evidence that formation of the destruction complex is driven by protein liquid-liquid phase separation (LLPS) of Axin. An intrinsically disordered region in Axin plays an important role in driving its LLPS. Phase-separated Axin provides a scaffold for recruiting GSK3ß, CK1α, and ß-catenin. APC also undergoes LLPS in vitro and enhances the size and dynamics of Axin phase droplets. The LLPS-driven assembly of the destruction complex facilitates ß-catenin phosphorylation by GSK3ß and is critical for the regulation of ß-catenin protein stability and thus Wnt/ß-catenin signaling.

Noncanonical TGF-ß signaling leads to FBXO3-mediated degradation of ΔNp63α promoting breast cancer metastasis and poor clinical prognosis.

Transforming growth factor-ß (TGF-ß) signaling plays a critical role in promoting epithelial-to-mesenchymal transition (EMT), cell migration, invasion, and tumor metastasis. ΔNp63α, the major isoform of p63 protein expressed in epithelial cells, is a key transcriptional regulator of cell adhesion program and functions as a critical metastasis suppressor. It has been documented that the expression of ΔNp63α is tightly controlled by oncogenic signaling and is frequently reduced in advanced cancers. However, whether TGF-ß signaling regulates ΔNp63α expression in promoting metastasis is largely unclear. In this study, we demonstrate that activation of TGF-ß signaling leads to stabilization of E3 ubiquitin ligase FBXO3, which, in turn, targets ΔNp63α for proteasomal degradation in a Smad-independent but Erk-dependent manner. Knockdown of FBXO3 or restoration of ΔNp63α expression effectively rescues TGF-ß-induced EMT, cell motility, and tumor metastasis in vitro and in vivo. Furthermore, clinical analyses reveal a significant correlation among TGF-ß receptor I (TßRI), FBXO3, and p63 protein expression and that high expression of TßRI/FBXO3 and low expression of p63 are associated with poor recurrence-free survival (RFS). Together, these results demonstrate that FBXO3 facilitates ΔNp63α degradation to empower TGF-ß signaling in promoting tumor metastasis and that the TßRI-FBXO3-ΔNp63α axis is critically important in breast cancer development and clinical prognosis. This study suggests that FBXO3 may be a potential therapeutic target for advanced breast cancer treatment.

Positive feedback of SuFu negating protein 1 on Hedgehog signaling promotes colorectal tumor growth.

Hedgehog (Hh) signaling plays a critical role in embryogenesis and tissue homeostasis, and its deregulation has been associated with tumor growth. The tumor suppressor SuFu inhibits Hh signaling by preventing the nuclear translocation of Gli and suppressing cell proliferation. Regulation of SuFu activity and stability is key to controlling Hh signaling. Here, we unveil SuFu Negating Protein 1 (SNEP1) as a novel Hh target, that enhances the ubiquitination and proteasomal degradation of SuFu and thus promotes Hh signaling. We further show that the E3 ubiquitin ligase LNX1 plays a critical role in the SNEP1-mediated degradation of SuFu. Accordingly, SNEP1 promotes colorectal cancer (CRC) cell proliferation and tumor growth. High levels of SNEP1 are detected in CRC tissues and are well correlated with poor prognosis in CRC patients. Moreover, SNEP1 overexpression reduces sensitivity to anti-Hh inhibitor in CRC cells. Altogether, our findings demonstrate that SNEP1 acts as a novel feedback regulator of Hh signaling by destabilizing SuFu and promoting tumor growth and anti-Hh resistance.