Yun Zhu

WNT2B is canonically characterized as a secreted WNT-family ligand, which is transported to the extracellular space via the endoplasmic reticulum (ER)-Golgi pathway and binds to cell surface FZDs (frizzled class receptors) to trigger downstream signaling cascades. Here, we identify a previously unrecognized non-secretory intracellular function of WNT2B in impairing endosomal trafficking to inhibit macroautophagy/autophagy, as well as a non-canonical LC3B-II-dependent autophagic secretion mechanism for WNT2B. Specifically, the non-secretory intracellular pool of WNT2B via its conserved middle domain (MD) binds to the spectrin repeat domain (SRD) of WASHC5, competitively displacing WASHC1 and thereby disrupting WASH complex assembly and inhibiting WASHC1-mediated actin polymerization on early endosomes. This disruption impairs endosomal cargo trafficking, including the core autophagy protein ATG9A, leading to defective autophagy initiation and subsequent accumulation of pro-inflammatory and pro-fibrotic factors in fibroblasts. We validated this mechanism in vivo using a TNBS-induced mouse model of chronic colitis. Fibroblast-specific wnt2b deletion restores autophagy, reduces pro-inflammatory cytokine secretion, and ameliorates intestinal fibrosis. Consistently, in Crohn disease (CD) patient tissues, elevated WNT2B in fibrotic regions negatively correlates with autophagy activity, and positively correlates with pro-fibrotic phenotypes, and clinical disease severity. Moreover, we identify a novel LC3B-II-dependent autophagic secretion pathway for WNT2B, which is distinct from the conventional ER-to-Golgi-dependent protein secretion. Collectively, our study delineates a novel non-canonical WNT2B-WASH complex-ATG9A regulatory axis through which WNT2B impairs endosomal trafficking and disrupts autophagy, ultimately amplifying inflammation and fibrosis. This study suggests that WNT2B may serve as a promising therapeutic target for CD and autophagy-associated fibrotic disorders.Abbreviations: 3-MA: 3-methyladenine; AAV: adeno-associated virus; ACTA2: actin alpha 2, smooth muscle; ARPC2: actin related protein 2/3 complex subunit 2; ATG: autophagy related; CCN3: cellular communication network factor 3; CD: Crohn disease; CK666: 2-fluoro-N-[2-(2-methyl-1H-indol-3-yl)ethyl]benzamide; COL1A1: collagen type I alpha 1 chain; Co-IP: co-immunoprecipitation; CTNNB1: catenin beta 1; DBcAMP: dibutyryl cyclic adenosine monophosphate; DPT: dermatopontin; EEA1: early endosome antigen 1; EGFR: epidermal growth factor receptor; ELISA: enzyme-linked immunosorbent assay; ER: endoplasmic reticulum; ESCRT: endosomal sorting complexes required for transport; EV: extracellular vesicle; FRAP: fluorescence recovery after photobleaching; FL: full length; FZD: frizzled class receptor; GST: glutathione S-transferase; HIF: human intestinal fibroblast; HMGB1: high mobility group box 1; IKBKB: inhibitor of nuclear factor kappa B kinase subunit beta; IL6: interleukin 6; LDELS: LC3-dependent EV loading and secretion; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MD: middle domain; MEFs: mouse embryonic fibroblasts; MTOR: mechanistic target of rapamycin kinase; MVB: multivesicular body; NFKB: nuclear factor kappa B; NFKBIA: NFKB inhibitor alpha; PDCD6IP: programmed cell death 6 interacting protein; PLA: proximity ligation assay; RELA/p65: RELA proto-oncogene, NF-kB subunit; SAFB: scaffold attachment factor B; SES-CD: Simple Endoscopic Score for Crohn disease; SIM: super-resolution structured illumination microscopy; SMAD3: SMAD family member 3; SQSTM1/p62: sequestosome 1; SRD: spectrin repeat domain; TEM: transmission electron microscopy; TFRC: transferrin receptor; TGFB1: transforming growth factor beta 1; TGOLN2: trans-golgi network protein 2; TNBS: 2,4,6-trinitrobenzenesulfonic acid; TNF: tumor necrosis factor; VCA: Verprolin homology, Central and Acidic; WASHC: WASH complex subunit; WLS: Wnt ligand secretion mediator; WCL: whole cell lysates; WNT: Wnt family member; WT, wild type.

Background: Icaritin, a natural compound extracted from Epimedium, has demonstrated efficacy and a favorable safety profile in treating hepatocellular carcinoma (HCC), providing an option for patients intolerant to conventional treatment. However, real-world data remain limited. To assess the therapeutic potential and safety of icaritin for patients with unresectable HCC (uHCC), we conducted a retrospective analysis at our center. Methods: This study analyzed 26 HCC patients treated with icaritin-based regimens (monotherapy, n = 10; combination therapy, n = 16) at Nanfang Hospital (June 2018–June 2024). We evaluated efficacy outcomes, including objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS), as well as safety and health-related quality of life (HRQOL). Results: The monotherapy group showed a DCR of 60.0%, median PFS of 3.5 months, and a median OS of 9.4 months. The combination therapy group had a DCR of 93.8%, a median PFS of 5.7 months, and a median OS of 10.6 months. The incidence of Grade 3 treatment-related adverse events (TRAEs) was 13.9%, and no Grade 4+ TRAEs were observed. HRQOL was maintained throughout treatment in either group. One patient with BCLC stage C achieved a partial response after 2 months of icaritin monotherapy and a complete response after 8 months, with PFS exceeding 18 months. Conclusion: Icaritin-based therapy has certain efficacy and a favorable safety in uHCC, suggesting a therapeutic alternative for uHCC patients ineligible for standard treatments.

Background: Cancer-associated fibroblasts (CAFs) and interleukins (ILs) family play crucial roles in pancreatic carcinoma (PC) immune response. However, the correlation between the IL family, CAFs infiltration, and PC prognosis remains uninvestigated. Methods: An IL family expression pattern for prognosis was constructed using a stepwise Cox proportional hazards regression model using TCGA data. Clinical data and validations from seven independent public cohort datasets were conducted to confirm the performance of the model. CAFs infiltrating abundance and spatial distribution in PC, and their correlation with patient prognosis were detected. Correlation between IL expression pattern, CAF infiltration, and immunotherapy response was evaluated using clinical tissue samples. Results: This study constructed the first IL family expression pattern to predict CAFs infiltration and prognosis in PC. The model was validated using clinical data and a meta-analysis of seven public PC datasets (HR= 1.27). IL high-risk patients had shorter survival, advanced tumors and lymph node metastasis compared to low-risk patients. Patients with unfavorable immunotherapy response had significantly higher IL risk scores (P=0.015). The IL expression pattern distinguished CAFs infiltration characteristics in PC, showing greater infiltration of CAFs, antigen-presenting CAFs (apCAFs) and inflammatory CAFs in the high-risk group. IL high-risk group also exhibited increased apCAF/tumor cell and apCAF/Tregs engagement, resulting in suppressed immune responses, crippled T-cell function and B-cell function, and elevated levels of biomarkers associated with poor immune response. Conclusion: This study constructed the first IL expression pattern related to CAFs infiltration, immunotherapy response, and prognosis in PC patients. This might promote precise immunotherapy and facilitate treatment options for PC.

Angiogenesis plays a vital role in colon cancer growth and metastasis. The role of protein kinase N2 (PKN2) in colon cancer is rarely studied. In this study, we investigated the effect of PKN2 on angiogenesis in colon cancer. We evaluated the correlation between PKN2 expression and microvessel density (MVD) in tumor tissue of patients with colon cancer. The effect of PKN2 on tumor angiogenesis was investigated both in cultured colon cancer cells and in a mouse colon cancer model. PKN2 targeted vascular endothelial growth factor A (VEGFA) and basic fibroblast growth factor (bFGF) expression, and secretion were analyzed, and the specific regulatory role of PKN2 on HIF was explored. PKN2 expression was negatively correlated with tumor MVD in tumor tissue of patients with colon cancer. PKN2 inhibited angiogenesis in both in vitro and in vivo models of mouse tumors. Mechanistically, PKN2 suppressed the transcriptional activity of hypoxia‐inducible factor‐1α (HIF‐1α) and reduced its nuclear accumulation, leading to the inhibiting of VEGFA and bFGF transcription by preventing HIF‐1α binding to their promoters. Additionally, PKN2 directly interacted with HIF‐1α at the protein level and induced phosphorylation, resulting in ubiquitination‐dependent degradation of HIF‐1α in colon cancer cells. Our study demonstrated, for the first time, that PKN2 exerts inhibitory effects on tumor angiogenesis in colon cancer. We propose a novel mechanism by which PKN2 regulates VEGFA and bFGF expression through modulation of the dynamic equilibrium of HIF‐1α protein levels.

Fibrosis is an important pathological change in inflammatory bowel disease (IBD), but the mechanism has yet to be elucidated. WNT2B high‑expressed fi

Background: New predictors of the efficacy of hepatocellular carcinoma (HCC) immunotherapy are needed. The ability of a single gene mutation to predict the therapeutic effect of immune checkpoint inhibitors (ICI) in HCC remains unknown. Methods: The most frequently mutated genes in HCC were analyzed using the Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) datasets. Mutant genes that correlated with the tumor mutational burden (TMB) and prognosis were obtained. The mutation pattern and immunological function of one of the most frequently mutated genes, LRP1B, were determined. A pan-tumor analysis of LRP1B expression, association with cancer prognosis, and immunological role was also explored. A retrospective clinical study was conducted using 102 HCC patients who received ICI treatment to further verify whether gene mutations can predict the effectiveness of immunotherapy and prognosis of HCC. Results: LRP1B is among the most frequently mutated genes in HCC cohorts in TCGA and ICGC datasets. TCGA data showed that the LRP1B mutation activated immune signaling pathways and promoted mast cell activation. Patients with LRP1B mutations had significantly higher TMB than those with wild-type LRP1B. LRP1B expression correlated with the cancer-immunity cycle and immune cell infiltration. High LRP1B expression was also associated with poor survival among HCC patients. Results from the clinical study showed that HCC patients in the LRP1B mutation group had a poor response to ICI and worse prognosis than the wild-type group. The LRP1B mutation group had significantly higher TMB and mast cell infiltration in tumor tissues. Conclusion: This study is the first to report that a single gene LRP1B mutation is associated with a poor clinical response to ICI treatment and negative outcomes in HCC patients. HighLRP1B expression correlated with tumor immunity and HCC prognosis.

Background: Accurate biomarkers to predict the prognosis and immunotherapy response in pancreatic carcinoma (PC) are needed. The interleukins (ILs) family members play crucial roles in immune cell activation. However, the expression patterns and clinical significance of IL family in PC needs further exploration. This study aimed to systematically investigate the expression and clinical significance of IL family in PC. Methods: Samples from TCGA PAAD dataset were used to analyze the expression pattern of IL family and to construct the prognosis predictor signature. The predictive value of the signature in prognosis was further explored in 90 clinical PC samples. Immune characteristics and the potential role in immunotherapy response prediction of the signature was evaluated in the clinical tissue samples using multiplex immunophenotyping technique and spatial analysis. The predictive value of the IL signature was then validated using data from five GEO datasets and one ICGC dataset. The characteristic immune profiles and predictive value of immunotherapy response of the signature were explored using data from TCGA PAAD dataset. Findings: An IL family expression based signature that can predict the prognosis in PC was constructed. Clinical PC samples analysis showed that the high-risk group is related to advanced tumor, lymph node metastasis, and high proliferation of tumor. IL family risk score was an independent predictor of prognosis in the clinical patients. The signature high-risk patients have low levels infiltration of T cells and cytotoxic T lmphocytes (CTLs) as well as high levels of Treg cells and antigen presenting cells (APCs) in the tumor samples. Moreover, the high-risk group had impaired CTL/tumor cells engagement, CTL/T helpler cells engagement and CTL/APC engagement in the tumor microenvironment. Analysis using TCGA dataset showed that these IL genes are enriched in proliferation, metastasis and chemoresistance pathways of PC. The signature high-risk patients were featured in suppressed immune response in TCGA dataset. Interpretation: This was the first IL family-based model for predicting prognosis and immunotherapy response for patients with PC. This might promote the precise application of immunotherapy and facilitate treatment options for PC patients. Funding Information: This work was supported by the National Natural Science Foundation of China (no. 82073174 &81802339) and Guangzhou Science and Technology plan project [No. 202102020185&202102020069]. Declaration of Interests: We declare no competing interests. Ethics Approval Statement: The study was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University.

Background. The specific functional roles of long noncoding RNAs (lncRNAs) as ceRNAs in colon cancer and their potential implications for colon cancer prognosis remain unclear. In the present study, a genome-wide analysis was performed to investigate the potential lncRNA-mediated ceRNA interplay in colon cancer based on the “ceRNA hypothesis.” The prognostic value of the lncRNAs was evaluated. Methods. A dysregulated lncRNA-associated ceRNA network was constructed based on the miRNA, lncRNA, and mRNA expression profiles in combination with the miRNA regulatory network by using an integrative computational method. Molecular biological techniques, including qPCR and gene knockdown techniques, were used to verify candidate targets in colon cancer. Survival analysis was performed to identify the candidate lncRNAs with prognostic value. Results. Our network analysis uncovered several novel lncRNAs as functional ceRNAs through crosstalk with miRNAs. The QRT-PCR assays of patient tissues as well as gene knockdown colon cancer cells confirmed the expression of top lncRNAs and their correlation with target genes in the ceRNA network. Functional enrichment analysis predicted that differentially expressed lncRNAs might participate in broad biological functions associated with tumor progression. Moreover, these lncRNAs may be involved in a range of cellular pathways, including the apoptosis, PI3K-AKT, and EGFR signaling pathways. The survival analysis showed that the expression level of several lncRNAs in the network was correlated with the prognosis of patients with colon cancer. Conclusions. This study uncovered a dysregulated lncRNA-associated ceRNA network in colon cancer. The function of the identified lncRNAs in colon cancer was preliminarily explored, and their potential prognostic value was evaluated. Our study demonstrated that lncRNAs could potentially serve as important regulators in the development and progression of colon cancer. Candidate prognostic lncRNA biomarkers in colon cancer were identified.