UBE2C-induced crosstalk between mono- and polyubiquitination of SNAT2 promotes lymphatic metastasis in bladder cancer.

Ubiquitination plays an essential role in protein stability, subcellular localization, and interactions. Crosstalk between different types of ubiquitination results in distinct biological outcomes for proteins. However, the role of ubiquitination-related crosstalk in lymph node (LN) metastasis and the key regulatory factors controlling this process have not been determined. Using high-throughput sequencing, we found that ubiquitin-conjugating enzyme E2 C (UBE2C) was overexpressed in bladder cancer (BCa) and was strongly associated with an unfavorable prognosis. Overexpression of UBE2C increased BCa lymphangiogenesis and promoted LN metastasis both in vitro and in vivo. Mechanistically, UBE2C mediated sodium-coupled neutral amino acid transporter 2 (SNAT2) monoubiquitination at lysine 59 to inhibit K63-linked polyubiquitination at lysine 33 of SNAT2. Crosstalk between monoubiquitination and K63-linked polyubiquitination increased SNAT2 membrane protein levels by suppressing epsin 1-mediated (EPN1-mediated) endocytosis. SNAT2 facilitated glutamine uptake and metabolism to promote VEGFC secretion, ultimately leading to lymphangiogenesis and LN metastasis in patients with BCa. Importantly, inhibition of UBE2C significantly attenuated BCa lymphangiogenesis in a patient-derived xenograft model. Our results reveal the mechanism by which UBE2C mediates crosstalk between the monoubiquitination and K63-linked polyubiquitination of SNAT2 to promote BCa metastasis and identify UBE2C as a promising target for treating LN-metastatic BCa.

PDGFRα+ITGA11+ fibroblasts foster early-stage cancer lymphovascular invasion and lymphatic metastasis via ITGA11-SELE interplay.

Cancer-associated fibroblasts (CAFs) exhibit considerable heterogeneity in advanced cancers; however, the functional annotation and mechanism of CAFs in early-stage cancers remain elusive. Utilizing single-cell RNA sequencing and spatial transcriptomic, we identify a previously unknown PDGFRα+ITGA11+ CAF subset in early-stage bladder cancer (BCa). Multicenter clinical analysis of a 910-case cohort confirms that PDGFRα+ITGA11+ CAFs are associated with lymphovascular invasion (LVI) and poor prognosis in early-stage BCa. These CAFs facilitate LVI and lymph node (LN) metastasis in early-stage BCa, as evidenced in a PDGFRα+ITGA11+ CAFs-specific deficient mouse model. Mechanistically, PDGFRα+ITGA11+ CAFs promote lymphangiogenesis via recognizing ITGA11 surface receptor SELE on lymphatic endothelial cells to activate SRC-p-VEGFR3-MAPK pathway. Further, CHI3L1 from PDGFRα+ITGA11+ CAFs aligns the surrounding matrix to assist cancer cell intravasation, fostering early-stage BCa LVI and LN metastasis. Collectively, our study reveals the crucial role of PDGFRα+ITGA11+ CAFs in shaping metastatic landscape, informing the treatment of early-stage BCa LVI.

SUMOylation-Driven mRNA Circularization Enhances Translation and Promotes Lymphatic Metastasis of Bladder Cancer.

Aberrant gene expression is a prominent feature of metastatic cancer. Translational initiation is a vital step in fine-tuning gene expression. Thus, exploring translation initiation regulators may identify therapeutic targets for preventing and treating metastasis. Herein, we identified that DHCR24 was overexpressed in lymph node (LN) metastatic bladder cancer and correlated with poor prognosis of patients. DHCR24 promoted lymphangiogenesis and LN metastasis of bladder cancer in vitro and in vivo. Mechanistically, DHCR24 mediated and recognized the SUMO2 modification at lysine 108 of hnRNPA2B1 to foster TBK1 mRNA circularization and eIF4F initiation complex assembly by enhancing hnRNPA2B1-eIF4G1 interaction. Moreover, DHCR24 directly anchored to TBK1 mRNA 3'-untranslated region to increase its stability, thus forming a feed forward loop to elevate TBK1 expression. TBK1 activated PI3K/Akt signaling to promote VEGFC secretion, resulting in lymphangiogenesis and LN metastasis. DHCR24 silencing significantly impeded bladder cancer lymphangiogenesis and lymphatic metastasis in a patient-derived xenograft model. Collectively, these findings elucidate DHCR24-mediated translation machinery that promotes lymphatic metastasis of bladder cancer and supports the potential application of DHCR24-targeted therapy for LN-metastatic bladder cancer. SIGNIFICANCE: DHCR24 is a SUMOylation regulator that controls translation initiation complex assembly and orchestrates TBK1 mRNA circularization to activate Akt/VEGFC signaling, which stimulates lymphangiogenesis and promotes lymph node metastasis in bladder cancer.

SUMOylation-triggered ALIX activation modulates extracellular vesicles circTLCD4-RWDD3 to promote lymphatic metastasis of non-small cell lung cancer.

Lymph node (LN) metastasis is one of the predominant metastatic routes of non-small cell lung cancer (NSCLC) and is considered as a leading cause for the unsatisfactory prognosis of patients. Although lymphangiogenesis is well-recognized as a crucial process in mediating LN metastasis, the regulatory mechanism involving lymphangiogenesis and LN metastasis in NSCLC remains unclear. In this study, we employed high-throughput sequencing to identify a novel circular RNA (circRNA), circTLCD4-RWDD3, which was significantly upregulated in extracellular vesicles (EVs) from LN metastatic NSCLC and was positively associated with deteriorated OS and DFS of patients with NSCLC from multicenter clinical cohort. Downregulating the expression of EV-packaged circTLCD4-RWDD3 inhibited lymphangiogenesis and LN metastasis of NSCLC both in vitro and in vivo. Mechanically, circTLCD4-RWDD3 physically interacted with hnRNPA2B1 and mediated the SUMO2 modification at K108 residue of hnRNPA2B1 by upregulating UBC9. Subsequently, circTLCD4-RWDD3-induced SUMOylated hnRNPA2B1 was recognized by the SUMO interaction motif (SIM) of ALIX and activated ALIX to recruit ESCRT-III, thereby facilitating the sorting of circTLCD4-RWDD3 into NSCLC cell-derived EVs. Moreover, EV-packaged circTLCD4-RWDD3 was internalized by lymphatic endothelial cells to activate the transcription of PROX1, resulting in the lymphangiogenesis and LN metastasis of NSCLC. Importantly, blocking EV-mediated transmission of circTLCD4-RWDD3 via mutating SIM in ALIX or K108 residue of hnRNPA2B1 inhibited the lymphangiogenesis and LN metastasis of NSCLC in vivo. Our findings reveal a precise mechanism underlying SUMOylated hnRNPA2B1-induced EV packaging of circTLCD4-RWDD3 in facilitating LN metastasis of NSCLC, suggesting that EV-packaged circTLCD4-RWDD3 could be a potential therapeutic target against LN metastatic NSCLC.

ZEB1-mediated biogenesis of circNIPBL sustains the metastasis of bladder cancer via Wnt/ß-catenin pathway.

BACKGROUND: Circular RNAs (circRNAs) circularized by back-splicing of pre-mRNA are widely expressed and affected the proliferation, invasion and metastasis of bladder cancer (BCa). However, the mechanism underlying circRNA biogenesis in mediating the distant metastasis of BCa still unexplored. METHODS: RNA sequencing data between BCa and normal adjacent tissues was applied to identify the differentially expressed circRNAs. The functions of circNIPBL in BCa were investigated via a series of biochemical experiments. The Clinical significance of circNIPBL was examined in a cohort of larger BCa tissues. RESULTS: In the present study, we identified a novel circRNA (hsa_circ_0001472), circNIPBL, which was significantly upregulated and had great influence on the poor prognosis of patients with BCa. Functionally, circNIPBL promotes BCa metastasis in vitro and in vivo. Mechanistically, circNIPBL upregulate the expression of Wnt5a and activated the Wnt/ß-catenin signaling pathway via directly sponged miR-16-2-3p, leading to the upregulation of ZEB1, which triggers the EMT of BCa. Moreover, we revealed that ZEB1 interacted with the flanking introns of exons 2-9 on NIPBL pre-mRNA to trigger circNIPBL biogenesis, thus forming a positive feedback loop. Importantly, circNIPBL overexpression significantly facilitated the distant metastasis of BCa in the orthotopic bladder cancer model, while silencing ZEB1 remarkably blocked the effects of metastasis induced by circNIPBL overexpression. CONCLUSIONS: Our study highlights that circNIPBL-induced Wnt signaling pathway activation triggers ZEB1-mediated circNIPBL biogenesis, which forms a positive feedback loop via the circNIPBL/miR-16-2-3p/Wnt5a/ZEB1 axis, supporting circNIPBL as a novel therapeutic target and potential biomarker for BCa patients.

An HGF-dependent positive feedback loop between bladder cancer cells and fibroblasts mediates lymphangiogenesis and lymphatic metastasis.

BACKGROUND: Cancer-associated fibroblasts (CAFs) play a vital role in facilitating tumor progression through extensive reciprocal interplay with cancer cells. Tumor-derived extracellular vesicles (EVs) are the critical mediators involved in the crosstalk between cancer cells and stromal cells, contributing to the metastasis of cancers. Yet, the biological mechanisms of tumor-derived EVs in triggering CAFs phenotype to stimulate the lymph node (LN) metastasis of bladder cancer (BCa) are largely unknown. Here, we aimed to explore the effects and molecular mechanisms of tumor-derived EV-mediated CAFs phenotype in regulating BCa LN metastasis. METHODS: The high-throughput sequencing was utilized to identify the crucial long non-coding RNA (lncRNA) associated with CAF enrichment in BCa. The functional role of the transition of fibroblasts to CAFs induced by LINC00665-mediated EVs was investigated through the in vitro and in vivo assays. Chromatin isolation by RNA purification assays, fluorescence resonance energy transfer assays, cytokine profiling and patient-derived xenograft (PDX) model were performed to explore the underlying mechanism of LINC00665 in the LN metastasis of BCa. RESULTS: We found that CAFs are widely enriched in the tumor microenvironment of BCa, which correlated with BCa lymphangiogenesis and LN metastasis. We then identified a CAF-associated long non-coding RNA, LINC00665, which acted as a crucial mediator of CAF infiltration in BCa. Clinically, LINC00665 was associated with LN metastasis and poor prognosis in patients with BCa. Mechanistically, LINC00665 transcriptionally upregulated RAB27B expression and induced H3K4me3 modification on the promoter of RAB27B through the recruitment of hnRNPL. Moreover, RAB27B-induced EVs secretion endowed fibroblasts with the CAF phenotype, which reciprocally induced LINC00665 overexpression to form a RAB27B-HGF-c-Myc positive feedback loop, enhancing the lymphangiogenesis and LN metastasis of BCa. Importantly, we demonstrated that blocking EV-transmitted LINC00665 or HGF broke this loop and impaired BCa lymphangiogenesis in a PDX model. CONCLUSION: Our study uncovers a precise mechanism that LINC00665 sustains BCa LN metastasis by inducing a RAB27B-HGF-c-Myc positive feedback loop between BCa cells and fibroblasts, suggesting that LINC00665 could be a promising therapeutic target for patients with LN metastatic BCa.

Epidemiology, treatments, and related biomarkers of locally advanced or metastatic urothelial carcinoma in Chinese population: A scoping review.

OBJECTIVE: Bladder cancer is the 13th most common cancer in China with the predominant histologic type being urothelial carcinoma (UC). Locally advanced and metastatic (la/m) UC accounts for 12% of UC and the five-year survival rate is only 39.4%, imposing a significant disease and economic burden on the patients. The aim of this scoping review is to synthesize existing evidence of epidemiology, the landscape of treatment options and associated efficacy and safety profiles, as well as treatment-related biomarkers among Chinese la/mUC patients. METHODS: A systematic search was conducted on five databases (PubMed, Web of Science, Embase, Wanfang, and CNKI) from January 2011 to March 2022 based on the scoping review criteria in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Extension for Scoping Reviews. RESULTS: A total of 6211 records were identified, and further review resulted in 41 relevant studies that met all criteria. Additional searches were conducted on epidemiology and treatment-related biomarkers of bladder cancer to supplement the evidence. Among 41 studies, 24 reported on platinum-based chemotherapy, eight on non-platinum-based chemotherapy, six on immunotherapy, two on targeted therapy, and one on surgery. Efficacy outcomes were summarized by line of therapy. Treatment-related biomarkers including PD-L1, HER2, and FGFR3 alterations were identified, and the alteration rate of FGFR3 of Chinese UC patients was lower than that of the western patients. CONCLUSIONS: Despite chemotherapy has been the main treatment choice for decades, appealing new therapeutic strategies including ICIs, targeted therapies and ADCs were applied in clinical practice. Further research on epidemiology and treatment-related biomarkers of la/mUC patients is needed given only a limited number of studies have been identified thus far. High genomic heterogeneity and complexity of molecular features were observed among la/mUC patients; thus, further studies are required to identify critical drivers and promote potential precise therapies.

Mutant KRAS Mediates circARFGEF2 Biogenesis to Promote Lymphatic Metastasis of Pancreatic Ductal Adenocarcinoma.

Circular RNAs (circRNA) contribute to cancer stemness, proliferation, and metastasis. The biogenesis of circRNAs can be impacted by the genetic landscape of tumors. Herein, we identified a novel circRNA, circARFGEF2 (hsa_circ_0060665), which was upregulated in KRASG12D pancreatic ductal adenocarcinoma (PDAC) and positively associated with KRASG12D PDAC lymph node (LN) metastasis. CircARFGEF2 overexpression significantly facilitated KRASG12D PDAC LN metastasis in vitro and in vivo. Mechanistically, circARFGEF2 biogenesis in KRASG12D PDAC was significantly activated by the alternative splicing factor QKI-5, which recruited U2AF35 to facilitate spliceosome assembly. QKI-5 bound the QKI binding motifs and neighboring reverse complement sequence in intron 3 and 6 of ARFGEF2 pre-mRNA to facilitate circARFGEF2 biogenesis. CircARFGEF2 sponged miR-1205 and promoted the activation of JAK2, which phosphorylated STAT3 to trigger KRASG12D PDAC lymphangiogenesis and LN metastasis. Importantly, circARFGEF2 silencing significantly inhibited LN metastasis in the KrasG12D/+Trp53R172H/+Pdx-1-Cre (KPC) mouse PDAC model. These findings provide insight into the mechanism and metastasis-promoting function of mutant KRAS-mediated circRNA biogenesis. SIGNIFICANCE: Increased splicing-mediated biogenesis of circARFGEF2 in KRAS-mutant pancreatic ductal adenocarcinoma activates JAK2-STAT3 signaling and triggers lymph node metastasis, suggesting circARFGEF2 could be a therapeutic target to inhibit pancreatic cancer progression.

CoQ10 alleviates hepatic ischemia reperfusion injury via inhibiting NLRP3 activity and promoting Tregs infiltration.

Hepatic ischemia-reperfusion injury (IRI) has been concerned as a main complication of liver surgery and transplantation. Previous studies show that reactive oxygen species (ROS) associated inflammation response and contribute to the liver damage during IRI. Coenzyme Q10 (CoQ10) has shown many beneficial effects on abrogating ROS production and ameliorating liver injury. This study found lower CoQ10 level in the process of liver IRI in a mouse model of hepatic IRI. Meanwhile, our results showed that CoQ10 administration significantly attenuate hepatic IRI proved by HE staining, serum ALT/AST. The NOD-like receptor protein 3 (NLRP3) inflammasome is activated by ROS which triggers the activation of inflammatory caspases. In this study, NLRP3 was significantly suppressed by CoQ10 while Foxp3 exhibited increased expression in liver. Furthermore, Kupffer cells (KCs) pretreated with CoQ10 under the condition of hypoxia and reoxygenation contributed to improved CD4+CD25+Foxp3+ regulatory T cells (Tregs) ratio in co-culture system. Furthermore, NLRP3 inflammasome activator treatment in vivo resulted in higher expression of caspase-1 and NLRP3 and reduction of Tregs in liver, which reversed the protection of CoQ10 in the liver injury. Taken together, our study discovered that CoQ10 can suppress NLRP3 activity in KCs and improves Foxp3+ Tregs differentiation depending on M2 macrophage polarization of KCs to ameliorate hepatic IRI.

A Prognostic Nomogram Based on Log Odds of Positive Lymph Nodes to Predict Overall Survival for Non-Metastatic Bladder Cancer Patients after Radical Cystectomy.

(1) Purpose: The purpose of this study was to evaluate the prognostic capacity of the pathological N status (pN), lymph node ratio (LNR), and the log odds of positive lymph nodes (LODDS), and to build a prognostic nomogram to predict overall survival (OS) for bladder cancer patients treated by radical cystectomy. (2) Methods: The clinical and pathological characteristics of 10,938 patients with bladder cancer were identified from the Surveillance, Epidemiology, and End Results (SEER) database from 2004 to 2017. The predictive capacity was assessed by univariate and multivariate Cox regression analyses, the area under the receiver operating characteristic curve (AUC), and C-index. Calibration curves, decision curve analysis (DCA), and risk-grouping were utilized to evaluate the predictive accuracy and discriminative ability of the nomogram. (3) Results: LODDS was an independent risk factor for bladder cancer (all p < 0.001) and demonstrated the highest values of C-index and AUC. The values of AUCs in the training cohort were 0.747, 0.743, and 0.735 for predicting 1-, 3-, and 5-year OS, respectively. Calibration curves and DCA curves suggested the excellent clinical application value of our nomogram. (4) Conclusions: LODDS is a better predictive indicator for bladder cancer patients compared to pN and LNR. The LODDS-incorporated nomogram has excellent accuracy and promising clinical application value for non-metastatic bladder cancer after radical cystectomy.

KRAS mutant-driven SUMOylation controls extracellular vesicle transmission to trigger lymphangiogenesis in pancreatic cancer.

Lymph node (LN) metastasis occurs frequently in pancreatic ductal adenocarcinoma (PDAC) and predicts poor prognosis for patients. The KRASG12D mutation confers an aggressive PDAC phenotype that is susceptible to lymphatic dissemination. However, the regulatory mechanism underlying KRASG12D mutation-driven LN metastasis in PDAC remains unclear. Herein, we found that PDAC with the KRASG12D mutation (KRASG12D PDAC) sustained extracellular vesicle-mediated (EV-mediated) transmission of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) in a SUMOylation-dependent manner and promoted lymphangiogenesis and LN metastasis in vitro and in vivo. Mechanistically, hnRNPA1 bound with SUMO2 at the lysine 113 residue via KRASG12D-induced hyperactivation of SUMOylation, which enabled its interaction with TSG101 to enhance hnRNPA1 packaging and transmission via EVs. Subsequently, SUMOylation induced EV-packaged-hnRNPA1 anchoring to the adenylate- and uridylate-rich elements of PROX1 in lymphatic endothelial cells, thus stabilizing PROX1 mRNA. Importantly, impeding SUMOylation of EV-packaged hnRNPA1 dramatically inhibited LN metastasis of KRASG12D PDAC in a genetically engineered KrasG12D/+ Trp53R172H/+ Pdx-1-Cre (KPC) mouse model. Our findings highlight the mechanism by which KRAS mutant-driven SUMOylation triggers EV-packaged hnRNPA1 transmission to promote lymphangiogenesis and LN metastasis, shedding light on the potential application of hnRNPA1 as a therapeutic target in patients with KRASG12D PDAC.

Aberrant Nuclear Export of circNCOR1 Underlies SMAD7-Mediated Lymph Node Metastasis of Bladder Cancer.

Circular RNAs (circRNA) containing retained introns are normally sequestered in the nucleus. Dysregulation of cellular homeostasis can drive their nuclear export, which may be involved in cancer metastasis. However, the mechanism underlying circRNA nuclear export and its role in lymph node (LN) metastasis of bladder cancer remain unclear. Here, we identify an intron-retained circRNA, circNCOR1, that is significantly downregulated in LN metastatic bladder cancer and is negatively associated with poor prognosis of patients. Overexpression of circNCOR1 inhibited lymphangiogenesis and LN metastasis of bladder cancer in vitro and in vivo. Nuclear circNCOR1 epigenetically promoted SMAD7 transcription by increasing heterogeneous nuclear ribonucleoprotein L (hnRNPL)-induced H3K9 acetylation in the SMAD7 promoter, leading to inhibition of the TGFß-SMAD signaling pathway. Nuclear retention of circNCOR1 was regulated by small ubiquitin-like modifier (SUMO)ylation of DDX39B, an essential regulatory factor responsible for circRNA nuclear-cytoplasmic transport. Reduced SUMO2 binding to DDX39B markedly increased circNCOR1 retention in the nucleus to suppress bladder cancer LN metastasis. By contrast, SUMOylated DDX39B activated nuclear export of circNCOR1, impairing the suppressive role of circNCOR1 on TGFß-SMAD cascade activation and bladder cancer LN metastasis. In patient-derived xenograft (PDX) models, overexpression of circNCOR1 and inhibition of TGFß signaling significantly repressed tumor growth and LN metastasis. This study highlights SUMOylation-induced nuclear export of circNCOR1 as a key event regulating TGFß-SMAD signaling and bladder cancer lymphangiogenesis, thus supporting circNCOR1 as a novel therapeutic agent for patients with LN metastatic bladder cancer. SIGNIFICANCE: This study identifies the novel intron-retained circNCOR1 and elucidates a SUMOylation-mediated DDX39B-circNCOR1-SMAD7 axis that regulates lymph node metastasis of bladder cancer.

Single-cell RNA sequencing reveals spatiotemporal heterogeneity and malignant progression in pancreatic neuroendocrine tumor.

Aims: Using Single-cell RNA sequencing (scRNA-seq), we explored the spatiotemporal heterogeneity of pancreatic neuroendocrine tumors (pNETs) and the underlying mechanism for malignant progression. Methods: scRNA-seq was conducted on three tumor tissues (two primary tissues from different sites, one liver metastatic lesion), one normal liver tissue, and peripheral blood mononuclear cells from one patient with a metastatic G2 pNET, followed by bioinformatics analysis and validation in a pNETs cohort. Results: The transcriptome data of 24.544 cells were obtained. We identified subpopulations of functional heterogeneity within malignant cells, immune cells, and fibroblasts. There were intra- and inter-heterogeneities of cell subpopulations for malignant cells, macrophages, T cells, and fibroblasts among all tumor sites. Cell trajectory analysis revealed several hallmarks of carcinogenesis, including the hypoxia pathway, metabolism reprogramming, and aggressive proliferation, which were activated at different stages of tumor progression. Evolutionary analysis based on mitochondrial mutations defined two dominant clones with metastatic capacity. Finally, we developed a gene signature (PCSK1 and SMOC1) defining the metastatic potential of the tumor and its prognostic value was validated in a cohort of thirty G1/G2 patients underwent surgical resection. Conclusions: Our scRNA-seq analysis revealed intra- and intertumor heterogeneities in cell populations, transcriptional states, and intercellular communications among primary and metastatic lesions of pNETs. The single-cell level characterization of the spatiotemporal dynamics of malignant cell progression provided new insights into the search for potential novel prognostic biomarkers of pNETs.

Tumor-derived exosomal BCYRN1 activates WNT5A/VEGF-C/VEGFR3 feedforward loop to drive lymphatic metastasis of bladder cancer.

BACKGROUND: Patients with lymph node (LN) metastatic bladder cancer (BCa) present with extremely poor prognosis. BCa-derived exosomes function as crucial bioactive cargo carriers to mediate the signal transduction in tumor microenvironment triggering tumor metastasis. However, the mechanisms underlying exosome-mediated LN metastasis in BCa are unclear. METHODS: We conducted the high-throughput sequencing to explore the expression profile of long noncoding RNA (lncRNA) in urinary exosomes (urinary-EXO) from patients with BCa and further evaluated the clinical relevance of exosomal lncRNA BCYRN1 in a larger 210-case cohort. The functional role of exosomal BCYRN1 was evaluated through the migration and tube formation assays in vitro and the footpad-popliteal LN metastasis model in vivo. RNA pull-down assays, luciferase assays, and actinomycin assays were conducted to detect the regulatory mechanism of exosomal BCYRN1. RESULTS: LncRNA BCYRN1 was substantially upregulated in urinary-EXO from patients with BCa, and associated with the LN metastasis of BCa. We demonstrated that exosomal BCYRN1 markedly promoted tube formation and migration of human lymphatic endothelial cells (HLECs) in vitro and lymphangiogenesis and LN metastasis of BCa in vivo. Mechanistically, BCYRN1 epigenetically upregulated WNT5A expression by inducing hnRNPA1-associated H3K4 trimethylation in WNT5A promoter, which activated Wnt/ß-catenin signaling to facilitate the secretion of VEGF-C in BCa. Moreover, exosomal BCYRN1 was transmitted to HLECs to stabilize the VEGFR3 mRNA and thus formed an hnRNPA1/WNT5A/VEGFR3 feedforward regulatory loop, ultimately promoting the lymphatic metastasis of BCa. Importantly, blocking VEGFR3 with specific inhibitor, SAR131675 significantly impaired exosomal BCYRN1-induced the LN metastasis in vivo. Clinically, exosomal BCYRN1 was positively associated with the shorter survival of BCa patients and identified as a poor prognostic factor of patients. CONCLUSION: Our results uncover a novel mechanism by which exosomal BCYRN1 synergistically enhances VEGF-C/VEGFR3 signaling-induced lymphatic metastasis of BCa, indicating that BCYRN1 may serve as an encouraging therapeutic target for patients with BCa.