Clinical significance of PNO1 as a novel biomarker and therapeutic target of hepatocellular carcinoma.

The RNA-binding protein PNO1 plays an essential role in ribosome biogenesis. Recent studies have shown that it is involved in tumorigenesis; however, its role in hepatocellular carcinoma (HCC) is not well understood. The purpose of this study was to examine whether PNO1 can be used as a biomarker of HCC and also examine the therapeutic potential of PNO1 knockout for the treatment of HCC. PNO1 expression was upregulated in HCC and associated with poor prognosis. PNO1 expression was positively associated with tumour stage, lymph node metastasis and poor survival. PNO1 expression was significantly higher in HCC compared to that in fibrolamellar carcinoma or normal tissues. Furthermore, HCC tissues with mutant Tp53 expressed higher PNO1 than those with wild-type Tp53. PNO1 knockout suppressed cell viability, colony formation and EMT of HCC cells. Since activation of Notch signalling pathway promotes HCC, we measured the effects of PNO1 knockout on the components of Notch pathway and its targets. PNO1 knockout suppressed Notch signalling by modulating the expression of Notch ligands and their receptors, and downstream targets. PNO1 knockout also inhibited genes involved in surface adhesion, cell cycle, inflammation and chemotaxis. PNO1 knockout also inhibited colony and spheroid formation, cell migration and invasion, and markers of stem cells, pluripotency and EMT in CSCs. Overall, our data suggest that PNO1 can be used as a diagnostic and prognostic biomarker of HCC, and knockout of PNO1 by CRISPR/Cas9 can be beneficial for the management of HCC by targeting CSCs.

A Notch toward Progenitor D(G)FRentiation: Defining a NOTCH-PDGFR axis in brown adipogenesis.

Brown adipocytes are found in several fat depots, however, the origins and contributions of different lineages of adipogenic progenitor cells (APCs) to these depots are unclear. In this issue of Developmental Cell, Shi et al. show that platelet-derived growth factor receptor ß (PDGFRß)-lineage and T-box transcription factor 18 (TBX18)-lineage APCs differentially contribute to brown adipogenesis across these depots.

The Clock and Wavefront Self-Organizing model recreates the dynamics of mouse somitogenesis in vivo and in vitro.

During mouse development, presomitic mesoderm cells synchronize Wnt and Notch oscillations, creating sequential phase waves that pattern somites. Traditional somitogenesis models attribute phase waves to a global modulation of the oscillation frequency. However, increasing evidence suggests that they could arise in a self-organizing manner. Here, we introduce the Sevilletor, a novel reaction-diffusion system that serves as a framework to compare different somitogenesis patterning hypotheses. Using this framework, we propose the Clock and Wavefront Self-Organizing model that considers an excitable self-organizing region where phase waves form independent of global frequency gradients. The model recapitulates the change in relative phase of Wnt and Notch observed during mouse somitogenesis and provides a theoretical basis for understanding the excitability of mouse presomitic mesoderm cells in vitro.

The people behind the papers - Julie Klepstad and Luciano Marcon.

The patterning of somites is coordinated by presomitic mesoderm cells through synchronised oscillations of Notch signalling, creating sequential waves of gene expression that propagate from the posterior to the anterior end of the tissue. In a new study, Klepstad and Marcon propose a new theoretical framework that recapitulates the dynamics of mouse somitogenesis observed in vivo and in vitro. To learn more about the story behind the paper, we caught up with first author Julie Klepstad and corresponding author Luciano Marcon, Principal Investigator at the Andalusian Center for Developmental Biology.

Jag1/2 maintain esophageal homeostasis and suppress foregut tumorigenesis by restricting the basal progenitor cell pool.

Basal progenitor cells are crucial for maintaining foregut (the esophagus and forestomach) homeostasis. When their function is dysregulated, it can promote inflammation and tumorigenesis. However, the mechanisms underlying these processes remain largely unclear. Here, we employ genetic mouse models to reveal that Jag1/2 regulate esophageal homeostasis and foregut tumorigenesis by modulating the function of basal progenitor cells. Deletion of Jag1/2 in mice disrupts esophageal and forestomach epithelial homeostasis. Mechanistically, Jag1/2 deficiency impairs activation of Notch signaling, leading to reduced squamous epithelial differentiation and expansion of basal progenitor cells. Moreover, Jag1/2 deficiency exacerbates the deoxycholic acid (DCA)-induced squamous epithelial injury and accelerates the initiation of squamous cell carcinoma (SCC) in the forestomach. Importantly, expression levels of JAG1/2 are lower in the early stages of human esophageal squamous cell carcinoma (ESCC) carcinogenesis. Collectively, our study demonstrates that Jag1/2 are important for maintaining esophageal and forestomach homeostasis and the onset of foregut SCC.

Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind.

Developmental programming involves the accurate conversion of signalling levels and dynamics to transcriptional outputs. The transcriptional relay in the Notch pathway relies on nuclear complexes containing the co-activator Mastermind (Mam). By tracking these complexes in real time, we reveal that they promote the formation of a dynamic transcription hub in Notch ON nuclei which concentrates key factors including the Mediator CDK module. The composition of the hub is labile and persists after Notch withdrawal conferring a memory that enables rapid reformation. Surprisingly, only a third of Notch ON hubs progress to a state with nascent transcription, which correlates with polymerase II and core Mediator recruitment. This probability is increased by a second signal. The discovery that target-gene transcription is probabilistic has far-reaching implications because it implies that stochastic differences in Notch pathway output can arise downstream of receptor activation.

To correctly give rise to future tissues, cells in an embryo must receive and respond to the right signals, at the right time, in the right way. This involves genes being switched on quickly, with cells often ensuring that a range of molecular actors physically come together at 'transcription hubs' in the nucleus ­ the compartment that houses genetic information. These hubs are thought to foster a microenvironment that facilitates the assembly of the machinery that will activate and copy the required genes into messenger RNA molecules. The resulting 'mRNAs' act as templates for producing the corresponding proteins, allowing cells to adequately respond to signals. For example, the activation at the cell surface of a molecule called Notch triggers a series of events that lead to important developmental genes being transcribed within minutes. This process involves a dedicated group of proteins, known as Notch nuclear complexes, quickly getting together in the nucleus and interacting with the transcriptional machinery. How they do this efficiently at the right gene locations is, however, still poorly understood. In particular, it remained unclear whether Notch nuclear complexes participate in the formation of transcription hubs, as well as how these influence mRNA production and the way cells 'remember' having been exposed to Notch activity. To investigate these questions, DeHaro-Arbona et al. genetically engineered fruit flies so that their Notch nuclear complexes and Notch target genes both carried visible tags that could be tracked in living cells in real time. Microscopy imaging of fly tissues revealed that, due to their characteristics, Notch complexes clustered with the transcription machinery and formed transcription hubs near their target genes. All cells exposed to Notch exhibited these hubs, but only a third produced the mRNAs associated with Notch target genes; adding a second signal (an insect hormone) significantly increased the proportion. This illustrates how 'chance' and collaboration influence the way the organism responds to Notch signalling. Finally, the experiments revealed that the hubs persisted for at least a day after removing the Notch signal. This 'molecular memory' led to cells responding faster when presented with Notch activity again. The work by DeHaro-Arbona sheds light on how individual cells respond to Notch signalling, and the factors that influence the activation of its target genes. This knowledge may prove useful when trying to better understand diseases in which this pathway is implicated, such as cancer.

Triptolide, a Cancer Cell Proliferation Inhibitor, Causes Zebrafish Muscle Defects by Regulating Notch and STAT3 Signaling Pathways.

Triptolide is a natural compound in herbal remedies with anti-inflammatory and anti-proliferative properties. We studied its effects on critical signaling processes within the cell, including Notch1 and STAT3 signaling. Our research showed that triptolide reduces cancer cell proliferation by decreasing the expression of downstream targets of these signals. The levels of each signal-related protein and mRNA were analyzed using Western blot and qPCR methods. Interestingly, inhibiting one signal with a single inhibitor alone did not significantly reduce cancer cell proliferation. Instead, MTT assays showed that the simultaneous inhibition of Notch1 and STAT3 signaling reduced cell proliferation. The effect of triptolide was similar to a combination treatment with inhibitors for both signals. When we conducted a study on the impact of triptolide on zebrafish larvae, we found that it inhibited muscle development and interfered with muscle cell proliferation, as evidenced by differences in the staining of myosin heavy chain and F-actin proteins in confocal fluorescence microscopy. Additionally, we noticed that inhibiting a single type of signaling did not lead to any significant muscle defects. This implies that triptolide obstructs multiple signals simultaneously, including Notch1 and STAT3, during muscle development. Chemotherapy is commonly used to treat cancer, but it may cause muscle loss due to drug-related adverse reactions or other complex mechanisms. Our study suggests that anticancer agents like triptolide, inhibiting essential signaling pathways including Notch1 and STAT3 signaling, may cause muscle atrophy through anti-proliferative activity.

Novel insight into FCSK-congenital disorder of glycosylation through a CRISPR-generated cell model.

BACKGROUND: FCSK-congenital disorder of glycosylation (FCSK-CDG) is a recently discovered rare autosomal recessive genetic disorder with defective fucosylation due to mutations in the fucokinase encoding gene, FCSK. Despite the essential role of fucokinase in the fucose salvage pathway and severe multisystem manifestations of FCSK-CDG patients, it is not elucidated which cells or which types of fucosylation are affected by its deficiency. METHODS: In this study, CRISPR/Cas9 was employed to construct an FCSK-CDG cell model and explore the molecular mechanisms of the disease by lectin flow cytometry and real-time PCR analyses. RESULTS: Comparison of cellular fucosylation by lectin flow cytometry in the created CRISPR/Cas9 FCSK knockout and the same unedited cell lines showed no significant change in the amount of cell surface fucosylated glycans, which is consistent with the only documented previous study on different cell types. It suggests a probable effect of this disease on secretory glycoproteins. Investigating O-fucosylation by analysis of the NOTCH3 gene expression as a potential target revealed a significant decrease in the FCSK knockout cells compared with the same unedited ones, proving the effect of fucokinase deficiency on EGF-like repeats O-fucosylation. CONCLUSION: This study expands insight into the FCSK-CDG molecular mechanism; to the best of our knowledge, it is the first research conducted to reveal a gene whose expression level alters due to this disease.

Reduction of specific enterocytes from loss of intestinal LGR4 improves lipid metabolism in mice.

Whether intestinal Leucine-rich repeat containing G-protein-coupled receptor 4 (LGR4) impacts nutrition absorption and energy homeostasis remains unknown. Here, we report that deficiency of Lgr4 (Lgr4iKO) in intestinal epithelium decreased the proportion of enterocytes selective for long-chain fatty acid absorption, leading to reduction in lipid absorption and subsequent improvement in lipid and glucose metabolism. Single-cell RNA sequencing demonstrates the heterogeneity of absorptive enterocytes, with a decrease in enterocytes selective for long-chain fatty acid-absorption and an increase in enterocytes selective for carbohydrate absorption in Lgr4iKO mice. Activation of Notch signaling and concurrent inhibition of Wnt signaling are observed in the transgenes. Associated with these alterations is the substantial reduction in lipid absorption. Decrement in lipid absorption renders Lgr4iKO mice resistant to high fat diet-induced obesity relevant to wild type littermates. Our study thus suggests that targeting intestinal LGR4 is a potential strategy for the intervention of obesity and liver steatosis.

The First Defined Null Allele of the Notch Regulator, a Suppressor of Deltex: Uncovering Its Novel Roles in Drosophila melanogaster Oogenesis.

Suppressor of deltex (Su(dx)) is a Drosophila melanogaster member of the NEDD4 family of the HECT domain E3 ubiquitin ligases. Su(dx) acts as a regulator of Notch endocytic trafficking, promoting Notch lysosomal degradation and the down-regulation of both ligand-dependent and ligand-independent signalling, the latter involving trafficking through the endocytic pathway and activation of the endo/lysosomal membrane. Mutations of Su(dx) result in developmental phenotypes in the Drosophila wing that reflect increased Notch signalling, leading to gaps in the specification of the wing veins, and Su(dx) functions to provide the developmental robustness of Notch activity to environmental temperature shifts. The full developmental functions of Su(dx) are unclear; however, this is due to a lack of a clearly defined null allele. Here we report the first defined null mutation of Su(dx), generated by P-element excision, which removes the complete open reading frame. We show that the mutation is recessive-viable, with the Notch gain of function phenotypes affecting wing vein and leg development. We further uncover new roles for Su(dx) in Drosophila oogenesis, where it regulates interfollicular stalk formation, egg chamber separation and germline cyst enwrapment by the follicle stem cells. Interestingly, while the null allele exhibited a gain in Notch activity during oogenesis, the previously described Su(dx)SP allele, which carries a seven amino acid in-frame deletion, displayed a Notch loss of function phenotypes and an increase in follicle stem cell turnover. This is despite both alleles displaying similar Notch gain of function in wing development. We attribute this unexpected context-dependent outcome of Su(dx)sp being due to the partial retention of function by the intact C2 and WW domain regions of the protein. Our results extend our understanding of the developmental role of Su(dx) in the tissue renewal and homeostasis of the Drosophila ovary and illustrate the importance of examining an allelic series of mutations to fully understand developmental functions.

Linarin Ameliorates Restenosis After Vascular Injury in Type 2 Diabetes Mellitus via Regulating ADAM10-Mediated Notch Signaling Pathway.

Vascular lesions frequently arise as complication in patients diagnosed with diabetes mellitus (DM). Presently, percutaneous coronary intervention (PCI) and antithrombotic therapy serve as primary treatments. However, in-stent restenosis persists as a challenging clinical issue following PCI, lacking sustained and effective treatment. Linarin (LN) exhibits diverse pharmacological activities and is regarded as a potential drug for treating various diseases, including DM. But its specific role in restenosis after vascular injury in DM patients remains unclear. A rat model of diabetes-related restenosis was established to evaluate the role of LN on neointimal hyperplasia. Vascular smooth muscle cells (VSMCs) stimulated by high glucose (HG, 30 mM) underwent LN treatment. Additionally, an overexpression plasmid of A disintegrin and metalloproteinases (ADAM10) was constructed to transfect VSMCs. We employed CCK-8, Brdu, wound-healing scratch, and transwell migration assays to evaluate the proliferation and migration of VSMCs. Furthermore, western blot and immunofluorescence assays were utilized to investigate the expressions of ADAM10 and the downstream Notch signaling pathway in vivo and in vitro models. LN notably alleviated intimal hyperplasia after vascular injury in DM rats and reduced the protein expression of ADAM10, alongside its downstream Notch1 signaling pathway-related proteins (Notch1, NICD and Hes1) in rat carotid artery tissues. LN effectively suppressed the proliferation and migration of VSMCs induced by HG, downregulating the protein expression of ADAM10, Notch1, NICD and Hes1. Moreover, our findings indicated that ADAM10 overexpression significantly reversed LN's effects on proliferation, migration, and the expression of Notch1 signaling pathway-related proteins in HG-treated VSMCs. LN demonstrates potential therapeutic efficacy in addressing restenosis after diabetic-related vascular injury, with the ADAM10 mediated Notch signaling pathway playing a pivotal role.

Osteoporosis: Emerging targets on the classical signaling pathways of bone formation.

Osteoporosis is a multifaceted skeletal disorder characterized by reduced bone mass and structural deterioration, posing a significant public health challenge, particularly in the elderly population. Treatment strategies for osteoporosis primarily focus on inhibiting bone resorption and promoting bone formation. However, the effectiveness and limitations of current therapeutic approaches underscore the need for innovative methods. This review explores emerging molecular targets within crucial signaling pathways, including wingless/integrated (WNT), bone morphogenetic protein (BMP), hedgehog (HH), and Notch signaling pathway, to understand their roles in osteogenesis regulation. The identification of crosstalk targets between these pathways further enhances our comprehension of the intricate bone metabolism cycle. In summary, unraveling the molecular complexity of osteoporosis provides insights into potential therapeutic targets beyond conventional methods, offering a promising avenue for the development of new anabolic drugs.

Molecular pathology of small cell lung cancer: Overview from studies on neuroendocrine differentiation regulated by ASCL1 and Notch signaling.

Pulmonary neuroendocrine (NE) cells are rare airway epithelial cells. The balance between Achaete-scute complex homolog 1 (ASCL1) and hairy and enhancer of split 1, one of the target molecules of the Notch signaling pathway, is crucial for NE differentiation. Small cell lung cancer (SCLC) is a highly aggressive lung tumor, characterized by rapid cell proliferation, a high metastatic potential, and the acquisition of resistance to treatment. The subtypes of SCLC are defined by the expression status of NE cell-lineage transcription factors, such as ASCL1, which roles are supported by SRY-box 2, insulinoma-associated protein 1, NK2 homeobox 1, and wingless-related integration site signaling. This network reinforces NE differentiation and may induce the characteristic morphology and chemosensitivity of SCLC. Notch signaling mediates cell-fate decisions, resulting in an NE to non-NE fate switch. The suppression of NE differentiation may change the histological type of SCLC to a non-SCLC morphology. In SCLC with NE differentiation, Notch signaling is typically inactive and genetically or epigenetically regulated. However, Notch signaling may be activated after chemotherapy, and, in concert with Yes-associated protein signaling and RE1-silencing transcription factor, suppresses NE differentiation, producing intratumor heterogeneity and chemoresistance. Accumulated information on the molecular mechanisms of SCLC will contribute to further advances in the control of this recalcitrant cancer.

Hypoxia preconditioning increases Notch1 activity by regulating DNA methylation in vitro and in vivo.

BACKGROUND: Our previous research has demonstrated that hypoxic preconditioning (HPC) can improve spatial learning and memory abilities in adult mice. Adult hippocampal neurogenesis has been associated with learning and memory. The Neurogenic locus notch homolog protein (Notch) was involved in adult hippocampal neurogenesis, as well as in learning and memory. It is currently unclear whether the Notch pathway regulates hippocampal neuroregeneration by modifying the DNA methylation status of the Notch gene following HPC. METHOD: The HPC animal model and cell model were established through repeated hypoxia exposure using mice and the mouse hippocampal neuronal cell line HT22. Step-down test was conducted on HPC mice. Real-time PCR and Western blot analysis were used to assess the mRNA and protein expression levels of Notch1 and hairy and enhancer of split1 (HES1). The presence of BrdU-positive cells and Notch1 expression in the hippocampal dental gyrus (DG) were examined with confocal microscopy. The methylation status of the Notch1 was analyzed using methylation-specific PCR (MS-PCR). HT22 cells were employed to elucidate the impact of HPC on Notch1 in vitro. RESULTS: HPC significantly improved the step-down test performance of mice with elevated levels of mRNA and protein expression of Notch1 and HES1 (P < 0.05). The intensities of the Notch1 signal in the control group, the H group and the HPC group were 2.62 ± 0.57 × 107, 2.87 ± 0.84 × 107, and 3.32 ± 0.14 × 107, respectively, and the number of BrdU (+) cells in the hippocampal DG were 1.83 ± 0.54, 3.71 ± 0.64, and 7.29 ± 0.68 respectively. Compared with that in C and H group, the intensity of the Notch1 signal and the number of BrdU (+) cells increased significantly in HPC group (P < 0.05). The methylation levels of the Notch1 promoter 0.82 ± 0.03, 0.65 ± 0.03, and 0.60 ± 0.02 in the C, H, and HPC groups, respectively. The methylation levels of Notch1 decreased significantly (P < 0.05). The effect of HPC on HT22 cells exhibited similarities to that observed in the hippocampus. CONCLUSION: HPC may confer neuroprotection by activating the Notch1 signaling pathway and regulating its methylation level, resulting in the regeneration of hippocampal neurons.

Tracing the origin of alveolar stem cells in lung repair and regeneration.

Alveolar type 2 (AT2) cells are stem cells of the alveolar epithelia. Previous genetic lineage tracing studies reported multiple cellular origins for AT2 cells after injury. However, conventional lineage tracing based on Cre-loxP has the limitation of non-specific labeling. Here, we introduced a dual recombinase-mediated intersectional genetic lineage tracing approach, enabling precise investigation of AT2 cellular origins during lung homeostasis, injury, and repair. We found AT1 cells, being terminally differentiated, did not contribute to AT2 cells after lung injury and repair. Distinctive yet simultaneous labeling of club cells, bronchioalveolar stem cells (BASCs), and existing AT2 cells revealed the exact contribution of each to AT2 cells post-injury. Mechanistically, Notch signaling inhibition promotes BASCs but impairs club cells' ability to generate AT2 cells during lung repair. This intersectional genetic lineage tracing strategy with enhanced precision allowed us to elucidate the physiological role of various epithelial cell types in alveolar regeneration following injury.

Targeting Wnt/ß-catenin signaling and its interplay with TGF-ß and Notch signaling pathways for the treatment of chronic wounds.

Wound healing is a tightly regulated process that ensures tissue repair and normal function following injury. It is modulated by activation of pathways such as the transforming growth factor-beta (TGF-ß), Notch, and Wnt/ß-catenin signaling pathways. Dysregulation of this process causes poor wound healing, which leads to tissue fibrosis and ulcerative wounds. The Wnt/ß-catenin pathway is involved in all phases of wound healing, primarily in the proliferative phase for formation of granulation tissue. This review focuses on the role of the Wnt/ß-catenin signaling pathway in wound healing, and its transcriptional regulation of target genes. The crosstalk between Wnt/ß-catenin, Notch, and the TGF-ß signaling pathways, as well as the deregulation of Wnt/ß-catenin signaling in chronic wounds are also considered, with a special focus on diabetic ulcers. Lastly, we discuss current and prospective therapies for chronic wounds, with a primary focus on strategies that target the Wnt/ß-catenin signaling pathway such as photobiomodulation for healing diabetic ulcers.

Mechanism of Notch Signaling Pathway in Malignant Progression of Glioblastoma and Targeted Therapy.

Glioblastoma multiforme (GBM) is the most aggressive form of glioma and the most common primary tumor of the central nervous system. Despite significant advances in clinical management strategies and diagnostic techniques for GBM in recent years, it remains a fatal disease. The current standard of care includes surgery, radiation, and chemotherapy, but the five-year survival rate for patients is less than 5%. The search for a more precise diagnosis and earlier intervention remains a critical and urgent challenge in clinical practice. The Notch signaling pathway is a critical signaling system that has been extensively studied in the malignant progression of glioblastoma. This highly conserved signaling cascade is central to a variety of biological processes, including growth, proliferation, self-renewal, migration, apoptosis, and metabolism. In GBM, accumulating data suggest that the Notch signaling pathway is hyperactive and contributes to GBM initiation, progression, and treatment resistance. This review summarizes the biological functions and molecular mechanisms of the Notch signaling pathway in GBM, as well as some clinical advances targeting the Notch signaling pathway in cancer and glioblastoma, highlighting its potential as a focus for novel therapeutic strategies.

Differentiation of intestinal stem cells toward goblet cells under systemic iron overload stress are associated with inhibition of Notch signaling pathway and ferroptosis.

Iron overload can lead to oxidative stress and intestinal damage and happens frequently during blood transfusions and iron supplementation. However, how iron overload influences intestinal mucosa remains unknown. Here, the aim of current study was to investigate the effects of iron overload on the proliferation and differentiation of intestinal stem cells (ISCs). An iron overload mouse model was established by intraperitoneal injection of 120 mg/kg body weight iron dextran once a fortnight for a duration of 12 weeks, and an iron overload enteroid model was produced by treatment with 3 mM or 10 mM of ferric ammonium citrate for 24 h. We found that iron overload caused damage to intestinal morphology with a 64 % reduction in villus height/crypt depth ratio, and microvilli injury in the duodenum. Iron overload mediated epithelial function by inhibiting the expression of nutrient transporters and enhancing the expression of secretory factors in the duodenum. Meanwhile, iron overload inhibited the proliferation of ISCs and regulated their differentiation into secretory mature cells, such as goblet cells, through inhibiting Notch signaling pathway both in mice and enteroid. Furthermore, iron overload caused oxidative stress and ferroptosis in intestinal epithelial cells. In addition, ferroptosis could also inhibit Notch signaling pathway, and affected the proliferation and differentiation of ISCs. These findings reveal the regulatory role of iron overload on the proliferation and differentiation of ISCs, providing a new insight into the internal mechanism of iron overload affecting intestinal health, and offering important theoretical basis for the scientific application of iron nutrition regulation.

Regulation of Notch signaling by non-muscle myosin II Zipper in Drosophila.

The Notch pathway is an evolutionarily conserved signaling system that is intricately regulated at multiple levels and it influences different aspects of development. In an effort to identify novel components involved in Notch signaling and its regulation, we carried out protein interaction screens which identified non-muscle myosin II Zipper (Zip) as an interacting partner of Notch. Physical interaction between Notch and Zip was further validated by co-immunoprecipitation studies. Immunocytochemical analyses revealed that Notch and Zip co-localize within same cytoplasmic compartment. Different alleles of zip also showed strong genetic interactions with Notch pathway components. Downregulation of Zip resulted in wing phenotypes that were reminiscent of Notch loss-of-function phenotypes and a perturbed expression of Notch downstream targets, Cut and Deadpan. Further, synergistic interaction between Notch and Zip resulted in highly ectopic expression of these Notch targets. Activated Notch-induced tumorous phenotype of larval tissues was enhanced by over-expression of Zip. Notch-Zip synergy resulted in the activation of JNK pathway that consequently lead to MMP activation and proliferation. Taken together, our results suggest that Zip may play an important role in regulation of Notch signaling.

Prognostic value of CD8+T cells related genes and exhaustion regulation of Notch signaling pathway in hepatocellular carcinoma.

Immunotherapy has emerged as the primary treatment modality for patients with advanced Hepatocellular carcinoma (HCC). However, its clinical efficacy remains limited, benefiting only a subset of patients, while most exhibit immune tolerance and face a grim prognosis. The infiltration of immune cells plays a pivotal role in tumor initiation and progression. In this study, we conducted an analysis of immune cell infiltration patterns in HCC patients and observed a substantial proportion of CD8+T cells. Leveraging the weighted gene co-expression network analysis (WGCNA), we identified 235 genes associated with CD8+T cell and constructed a risk prediction model. In this model, HCC patients were stratified into a high-risk and low-risk group. Patients in the high-risk group exhibited a lower survival rate, predominantly presented with intermediate to advanced stages of cancer, displayed compromised immune function, showed limited responsiveness to immunotherapy, and demonstrated elevated expression levels of the Notch signaling pathway. Further examination of clinical samples demonstrated an upregulation of the Notch1+CD8+T cell exhaustion phenotype accompanied by impaired cytotoxicity and cytokine secretion functions that worsened with increasing Notch activation levels. Our study not only presents a prognostic model but also highlights the crucial involvement of the Notch pathway in CD8+T cell exhaustion-a potential target for future immunotherapeutic interventions.