SLeX decorated integrin α3 on sEVs promotes metastasis of bladder cancer via enhancing vascular permeability

doi:10.21203/rs.3.rs-4389823/v1

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SLeX decorated integrin α3 on sEVs promotes metastasis of bladder cancer via enhancing vascular permeability

https://doi.org/10.21203/rs.3.rs-4389823/v1

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published 02 Sep, 2024

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The permeability of blood vessels plays a crucial role in the spread of cancer cells, leading to their metastasis at distant sites. Small extracellular vesicles (sEVs) contribute to the metastasis of various cancers by crossing the blood vessel wall. However, the role of abnormal glycoconjugates on sEVs in tumor blood vessels is unknown. Our study found elevated levels of fucosyltransferase VII and its product sialyl Lewis X (sLeX) in muscle-invasive bladder cancer (BLCA), and high levels of sLeX can promote growth and invasion of BLCA cells. Further study revealed that sLeX was enriched in sEVs originating from BLCA. sLeX-decorated sEVs increased blood vessel permeability by disrupting the tight junctions of human umbilical vein endothelial cells (HUVECs). Using a glycoproteomics approach, we identified integrin α3 (ITGA3) as a sLeX-bearing glycoprotein in BLCA cells and their sEVs. Mechanically, sLeX modification stabilized ITGA3 by inhibiting its degradation in lysosomes. sEVs carrying sLeX-modified ITGA3 can be effectively internalized by HUVECs, leading to decreased expression of tight junction protein. In contrast, silencing ITGA3 in sLeX-decorated sEVs restored tight junction protein and reduced blood vessel permeability by inhibiting the MAPK pathway. Moreover, ITGA3 sLeX-modification at Asn 265 in HUVECs promoted occludin dephosphorylation on Ser/Thr residues, followed by inducing its importin α1-mediated nuclear translocation and resulting destroyed tight junction. Our findings suggest a potential strategy for disrupting the formation of a metastatic microenvironment and preventing the spread of malignant bladder cancer.

Sialyl Lewis X

sEVs

vascular permeability

metastasis

bladder cancer

Despite recent advances in cancer treatment, metastasis continues to a major contributor to cancer-related deaths. The process of metastasis involves the invasion of tumor cells into blood vessels or lymphatics, followed by their migration to secondary or distant organ sites. Recent studies have revealed that small extracellular vesicles (sEVs) play a role in the intravasation and extravasation of metastatic tumor cells [1, 2]

sEVs, characterized by nanometer-sized vesicles (30–150 nm diameter) secreted by cells, participate in various biological processes such as angiogenesis and metastasis [3–5], immune system modulation [6], and pre-metastatic niche formation [7], et al. They contain bioactive components like proteins, messenger RNA, microRNAs, and long non-coding RNA. sEVs have been shown to deliver oncogenic miRNAs or proteins, thereby increasing vascular permeability and promoting tumor metastasis [8–11]. Notably, the surface of sEVs is heavily adorned with glycoconjugates such as proteoglycans, glycosphingolipids, and glycoproteins [12–15]. These glycoconjugates play fundamental roles in vesicular protein sorting and sEVs uptake [16, 17]. However, the specific role of these glycoconjugates in tumor metastasis and their impact on vascular permeability remain to be fully explored.

Our previous study revealed elevated expression of sialyl Lewis X (sLeX, NeuAcα2-3Galβ1–4 [Fucα1–3] GlcNAcβ1-3Galβ1-R) in bladder cancer (BLCA) cells through lectin array and mass-spectrum analysis [18]. As a carbohydrate tumor marker, sLeX has been linked to tumor metastasis and poor patient outcomes across various tumor types [19, 20]. Functionally, sLeX acts as an adhesion ligand for E-selectin on leukocytes and stimulates vascular endothelial cells, thereby promoting the extravasation of leukocytes into inflamed tissues [21]. Studies have demonstrated that colon cancer cells expressing high level of sLeX display increased invasiveness and a higher propensity for metastasis. This effect can be attenuated by blocking the interaction between sLeX and E-selectin [22]. However, the distribution of sLeX on sEVs and its impact on their function remain unclear.

In the current study, we observed an enrichment of sLeX on sEVs secreted by BLCA cells and investigated the role of sLeX-decorated sEVs in BLCA metastasis by examining their effects on the expression and subcellular localization of tight junction (TJ)-related proteins and their promotion of vascular permeability.

Patient samples

Plasma of normal subjects and BLCA patients were obtained from the First Affiliated Hospital of Xi’an Jiaotong University and the Shaanxi Provincial People's Hospital. Written informed consent was obtained from all patients in accordance with the Declaration of Helsinki. Experiments using human samples were approved by the Research Ethics Committee of Northwest University.

Identification of proteins with sLeX

Proteins (500 µg) were denatured with lysate buffer (8 M urea and 50 mM NH₄HCO₃), followed by centrifugation at 10,000g for 15 min at room temperature. The supernatant was reduced by DTT and alkylated by IAM. The protein solution was diluted with deionized water to decrease urea concentration below 1 M, and digested with Lys-C (Wako Puro Chemical; Osaka, Japan) and trypsin (Promega; Madison, WI, USA). Samples were acidified with 10% trifluoroacetic acid to pH < 2 and purified using Oasis MAX cartridges and Oasis HLB cartridges, and subjected to two-dimensional LC-MS analysis using LTQ Orbitrap MS (Thermo Fisher). Proteomics data were analyzed by MaxQuant software [23], and glycoproteomics data were analyzed via Glyco-Decipher-v1.0.2 software program [24].

Tissue microarray/immunohistochemical analysis

BLCA tissue microarray (TMA) containing 30 cases of bladder carcinoma tissue with matched adjacent non-carcinoma tissue were from Shanghai Outdo Biotech Co. Tissue sections were deparaffinized and rehydrated as previous described [25]. Tissue sections were ordinally incubated with primary antibodies and corresponding HRP-labeled secondary antibodies. TMA was further stained with 3, 3’-diaminobenzidine (DAB) and observed under microscope. The images were quantitatively analyzed using Image Pro Plus software.

Animal models

Animal experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of Northwest University. BALB/c nude mice (4–5 weeks old) were purchased from Vital River Laboratory Animal Technology (Beijing, China). A total of 1×10⁶ cells in 100 µL of PBS were subcutaneously injected into the right dorsal side of mice. Tumor images were observed for 21 days post-xenografting.

For the footpad model, 2×10⁶ cells suspended in 50 µl PBS were injected into the footpad of the mice. After 21 days, the size of the footpad tumors and the popliteal lymph nodes (LNs) was measured, followed by fixation and embedding of the specimens in paraffin for immunohistochemical staining.

Vascular permeability assay in vivo was described as previously [26]. In brief, 20 µg sEVs^serum from healthy individuals and BLCA patients were intravenously injected into the tail vein of mice for once injection every other day. After a two-week period, mice received an intravenous injection of 100 mg/kg FITC-dextran. One hour post-injection, the presence of FITC-dextran in the ear vessel bloodstream and any leakage from the vasculature were examined using a laser confocal scanning microscope. Subsequently, mice were euthanized, and lung tissues were collected, embedded, and prepared as frozen blocks for sectioning and subsequent immunofluorescent staining.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 8.0 (GraphPad software, USA). The results are presented as the mean ± standard deviation (SD) from three or more independent experiments. Statistical comparisons between two groups were determined using an unpaired two-tailed Student's t-test. One-way ANOVA with Tukey’s multiple comparisons test was used for comparisons between multiple groups. The Mantel Cox test was employed to evaluate the correlation between the test index and survival of BLCA patients in TMA. P < 0.05 was considered as statistically significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not statistically significant

Expression of FUT7 and sLeX was positively associated with BLCA metastasis

It is known that fucosyltransferase VII (FUT7) is the key rate-limiting enzyme for sLeX synthesis (Fig. 1A) [27]. Using the TCGA database, we observed that FUT7 expression was elevated in various tumor types with high neoplasm histologic grades (Fig. S1A), and was associated with advanced clinical T stages (Fig. 1B), correlating with poor survival outcomes in BLCA patients (Fig. 1C). TMA analysis confirmed that FUT7 expression was significantly higher in BLCA tissues compared to normal adjacent tissues (Fig. 1D; Fig. S1B, C).

Consistent with abnormal FUT7 expression, higher level of sLeX were observed in BLCA tissues compared to normal tissues (Fig. 1E; Fig. S1B, D). The serum from BLCA patients also presented elevated level of sLeX compared to healthy controls (Fig. S1E). Moreover, high expression of FUT7 and elevated level of sLeX were closely correlated with lymph node metastasis, lymphovascular metastasis (Fig. 1F, G; Fig. S1F-H) and were indication of poor survival outcomes for BLCA patients (Fig. 1H, I).

SLeX promoted the malignant progression of BLCA

FUT7 expression and sLeX level were elevated in muscle-invasive BLCA cell lines (J82, T24 and YTS-1) compared to non-muscle-invasive BLCA cell lines (RT-4, KK47 and 5637) and human bladder epithelial cell lines (HCV29) (Fig. S2A-C). Introduction of the FUT7 gene into KK47 cells (referred to as oeFUT7) resulted in a significant increase in sLeX level (Fig. 2A), and enhanced the migration and invasion capabilities of the cells (Fig. 2B, C). Additionally, high level of sLeX was observed to stimulate cell colony formation and proliferation in KK47 cells (Fig. 2D, E). On the other hand, silencing of FUT7 expression in YTS-1 cells (referred to as shFUT7) significantly decreased sLeX level (Fig. 2A), and suppressed cell migration (Fig. 2B, C), colony formation and proliferation (Fig. 2F, G). However, no significant alteration of apoptosis was noted in either shFUT7 or oeFUT7 (Fig. S2D).

The mouse xenograft model results showed that tumorigenic rate was significantly higher in the oeFUT7 group compared to the KK47 group (Fig. S2E). In the footpad model, mice injected with oeFUT7 cells demonstrated significantly increased metastasis to the popliteal LNs compared to those injected with KK47 cells (Fig. S2F, G). The oeFUT7 group displayed larger weight and size of the footpad tumors and popliteal LNs (Fig. S2H). These in vitro and in vivo findings indicate that high level of sLeX promote bladder tumor growth and invasion.

BLCA derived-sEVs increased vascular permeability in vivo and in vitro

The metastasis of malignant carcinoma is largely driven by increased permeability of vascular endothelial cells (ECs) in tumor microenvironment. Our observation showed that the conditioned medium (CM) from YTS-1 cells significantly compromised the integrity of monolayer endothelial cell (Fig. S3A and B). Recognizing that sEVs play a crucial role as components of CM from malignant tumors and facilitate metastasis by disrupting the vascular barriers [8, 26, 28], we next isolated the sEVs from HCV29, KK47 and YTS-1 cells, and confirmed their sphere-like morphology (Fig. 3A), a narrow size distribution around 100 nm (Fig. 3B) and robust expression of sEV markers such as CD63, Alix, and Tsg101 (Fig. 3C).

Trans-endothelial migration assay showed that a higher number of GFP-labeled KK47 cells migrated through sEVs^YTS−1 treated HUVEC monolayers compared to sEVs^KK47 or sEVs^HCV29 treated group (Fig. 3D, E). Similarly, HUVEC monolayers treated with sEVs^YTS−1 showed increased permeability to rhodamine (Fig. 3D, F) and exhibited enhanced tube formation compared to the other two groups (Fig. S3C). It is known that the permeability of endothelial layer mainly depends on cell-cell junctions [29]. We then examined the transmembrane adhesive proteins in aforementioned HUVECs. TJ-associated proteins ZO-1, occludin and claudin-5 were significantly downregulated in the CM from YTS-1 and in the sEVs^YTS−1-treated groups, but not in the other groups, while adhesion junction (AJ)-associated proteins such as E-Cadherin, N-Cadherin, and β-catenin did not show a clear difference in all groups (Fig. 3G; S3D and E). In contrast, the treatment with GW4869, a widely-utilized sEVs biogenesis inhibitor, restored TJ proteins expression in CM treated-HUVECs (Fig. S3F). In vivo mouse study revealed that injection with sEVs^serum from BLCA patients resulted in the disruption of vascular barrier, indicated as the leakage of FITC-dextran out of blood vessels and more fluorescence in lung section, compared to the injection with sEVs^serum from healthy controls (Fig. 3H, I). Not surprisingly, sEVs^YTS−1 showed higher level of sLeX than sEVs^HCV29 and sEVs^KK47 (Fig. 3J). Elevated level of sLeX was also presented in sEVs derived from the serum of BLCA patients (Fig. 3K).

SLeX modified-vesicles were efficiently uptaken by HUVECs and destroyed vascular endothelial barriers

To assess the impact of sLeX modification on sEVs, we isolated sEVs from KK47 and oeFUT7 cells (referred to as sEVs^KK47 and sEVs^oeFUT7, respectively). sEVs^oeFUT7 presented a higher level of sLeX modification compared to sEVs^KK47 (Fig. 4A; Fig. S4A). Moreover, sEVs^oeFUT7 demonstrated more efficient internalization by HUVECs (Fig. 4B). Given that sLeX acts as a ligand for E-selectin on inflammatory endothelial cells [30], we pre-treated the HUVECs with the E-selectin inhibitor, A-205804 (Fig. S4B), resulting in reduced endocytosis of sEVs^oeFUT7 (Fig. 4C).

The expression of TJ proteins, including ZO-1, occludin, and claudin-5, were diminished in HUVECs treated with CM^oeFUT7 or sEVs^oeFUT7 compared to those treated with CM^KK47 or sEVs^KK47 (Fig. 4D; Fig. S4C, D). The permeability of HUVECs increased to both rhodamine and GFP⁺ KK47 cells in the sEVs^oeFUT7-pretreated group compared to the sEVs^KK47-pretreated group (Fig. 4E, F). Conversely, TJ proteins were elevated, and permeability reduced in HUVECs treated with CM^shFUT7 or sEVs^shFUT7 compared to those treated with CM^YTS−1 or sEVs^YTS−1 (Fig. S4C; Fig. 4G-I). Tube formation was increased in HUVECs treated with CM^oeFUT7 (compared to CM^KK47), but abolished in HUVECs treated with CM^shFUT7 (compared to CM^YTS−1) (Fig. S4E). Furthermore, Spearman correlation analysis demonstrated that FUT7 expression was positively correlated with angiogenesis pathway (Fig. S4F).

The above results were further validated by the in vivo oeFUT7-injected mouse footpad model. Compared to the control group, oeFUT7-injected group showed lower density of vessel integrity, indicated by occludin and claudin-5, and higher density of neovascularization, indicated by VEGF and CD31, in both the peritumoral and intratumoral regions (Fig. 4J, K). This suggests that high sLeX expression significantly disrupts vascular integrity and induces neovascular formation to facilitate tumor metastasis.

Vesicular ITGA3 was identified as a target protein modified by sLeX

It is known that sLeX are terminal epitopes of N/O-linked glycans on proteins and of glycolipids. Using proteomic analysis, we identified 3,654 differentially expressed proteins including 299 downregulated and 483 upregulated proteins, in response to altered sLeX modification in KK47 Vs. oeFUT7 cells (Fig. 5A; Fig. S5A; Table S2). The upregulated proteins were primarily enriched in cellular processes such as mitotic vasculogenesis, tight junction assembly, cell-cell adhesion, and integrin binding (Fig. 5B; Table S3), indicating that sLeX modification was closely associated with cell-cell adhesions. Further antibody based enrichment and mass spectrum (MS)-based glycoproteomic analysis identified integrin α3 (referred to as ITGA3) as a sLeX-bearing glycoprotein (Fig. 5C, D; Fig. S5B; Table S4).

Immunoprecipitation (IP) confirmed the enhanced sLeX modification on ITGA3 in oeFUT7 cells and sEVs^oeFUT7 (Fig. 5E, F). A correlation was observed between higher ITGA3 expression and higher sLeX modification level (Fig. 5A; Fig. S5C, D). Immunofluorescence (IF) staining also revealed a marked raise of both sLeX and ITGA3 in oeFUT7 cells, but not in KK47 cells (Fig. S5E). Spearman correlation analysis further illuminated a positive correlation between FUT7 and ITGA3 expression (Fig. S5F), suggesting that sLeX modification regulates ITGA3 expression at the protein level. Inhibiting protein synthesis with cycloheximide (CHX) resulted in the slower degradation of ITGA3 in oeFUT7 relative to KK47 cells (Fig. S5G, H). Further study revealed that sLeX modification stabilizes ITGA3 by inhibiting its degradation via the lysosomal pathway (Fig. S5I, J).

ITGA3 promoted vascular permeability and angiogenesis

The IHC results of The Human Protein Altas (HPA) database showed that BLCA patients with high expression of ITGA3 account for approximately 75% of all BLCA patients (Fig. 6A; Fig. S6A; Table S5). ITGA3 in serum and serum-sEVs were clearly enhanced from BLCA patients than those from healthy individuals (Fig. 6B, C). Using TMA analysis, we found significant increases in ITGA3 expression in BLCA tissues than paired normal tissues (Fig. 6D, E; Fig. S6B, C). ITGA3 expression was also associated with lymphovascular metastasis, lymph node metastasis and poor prognosis in BLCA patients (Fig. 6F-H). Similarly, Spearman correlation analysis showed that the expression of ITGA3 was positively correlated with the angiogenesis in BLCA (Fig. 6I). Based on these findings, we hypothesize that sEVs enter HUVECs and disrupt vascular endothelial barriers through the highly sLeX-modified ITGA3.

As mentioned above, GO analysis indicated the correlation of ITGA3 expression with tight junction and angiogenesis. Spearman correlation analysis revealed that ITGA3 expression was positively correlated with VEGFA (vascular endothelial growth factor A), whereas negatively correlated with TJ-related proteins, including claudin-5, occludin, ESAM and JAM3 (Fig. 7A). When ITGA3 gene was stably overexpressed in HUVECs (referred to as oeITGA3), the proteins involved in the MAPK pathway in endothelial permeability [31, 32], including phosphorylated c-Jun and p38 were increased in oeITGA3 cells, along with reduced TJ protein expression and increased endothelial permeability (Fig. 7B, C). Consistent with these findings, incubation with CM^oeITGA3 promoted tube formation of HUVECs (Fig. S6D). These results demonstrate that ITGA3 can disrupt vascular endothelial barriers and induce angiogenesis by activating MAPK signaling.

Silencing ITGA3 reversed sLeX induced tumorigenesis and metastasis

After endocytosis, NHS-biotin-labelled vesicular ITGA3 of oeFUT7 can be recycled by HUVECs (Fig. 7C, D). The silence of ITGA3 in oeFUT7 cells (referred to as oe-shITGA3) resulted in the decreased ITGA3 expression in sEVs (Fig. S6E, F), but did not affect the endocytosis of sEVs into endothelial cells (Fig. S6G). The permeability of HUVECs was dramatically reduced when incubated with CM^oe−shITGA3 or sEVs^oe−shITGA3 compared with CM^oeFUT7 or sEVs^oeFUT7 (Fig. S6H, I; Fig. 7G, H), accompanied by the restoration of the expression of ZO-1, occludin and claudin-5 (Fig. 7F). The MAPK-associated proteins were activated in sEVs^oeFUT7-treated HUVECs, but deactivated in sEVs^oe−shITGA3-incubated HUVECs (Fig. 7F). Additionally, less tube formation was observed in HUVECs incubated with CM^oe−shITGA3 than CM^oeFUT7 (Fig. S6I).

Using popliteal LN metastasis model, we found that the high incidence of tumorigenic and LN metastasis events in oeFUT7 injected mice was significantly reversed by ITGA3 knockdown (Fig. S6K-M). ITGA3-knockdown significantly increased the expression of occludin and claudin-5, and decreased the density of VEGF and CD31-indicated neovascular in both peritumoral and intratumoral regions of primary footpad tumor (Fig. 7I, J). These findings collectively demonstrate that the silence of ITGA3 reversed the tumor growth, metastasis, and angiogenesis induced by sLeX-modified sEVs, and restored the paracancer endothelium barrier in vivo.

SLeX-modified ITGA3 induced occludin translocation into nuclear

As illustrated in Fig. 5D, asparagine 265 (Asn265) was identified as the primary sLeX-modified site of ITGA3. The site-specific mutant replacing Asn265 by Ala on ITGA3 (referred to as N265A-HA) showed the sharply reduced level of sLeX modification (Fig. 8A). The inhibition of TJ-related proteins was observed in ITGA3-HA HUVECs, accompanied by activation of the MAPK signaling pathway. However, this suppressive effect was significantly reversed in N265A-HA cells, concomitant with the inactivation of the MAPK signaling pathway (Fig. 8B, C). Similar to wild-type HUVECs, the permeability of HUVECs to rhodamine decreased in the N265A-HA group compared to the ITGA3-HA group (Fig. 8D). Additionally, we noted a reduction in the ability of tube formation in the N265A-HA group compared to the ITGA3-HA group (Fig. S7A).

Mechanically, Co-immunoprecipitation (Co-IP) coupled with tandem LC–MS/MS analysis compared the differential proteins interacting with ITGA3 in ITGA3-HA and N265A-HA cells. Of the 12 proteins presenting inhibited interaction with ITGA3 in N265A-HA group, PP1A was screened out; notably, PP1A has been shown to regulate the tight junctions between endothelial cells by dephosphorylating occludin at Ser/Thr residues [33] (Fig. 8E, F). Co-IP assays showed that sLeX-modified ITGA3 can assist occludin to interact with PP1A and result in dephosphorylation of occludin at Ser/Thr residues, whereas this effect was reversed in N265A-HA (Fig. 8G, H). Consistent with the observation that phosphorylation state of occludin at Ser/Thr residues can affect its subcellular localization [33–36], we found that more occludin entered the cytoplasm and nuclear in ITGA3-HA, while occludin predominantly remained at the cell membrane to maintain TJ between endothelial cells in N265A-HA (Fig. 8I, J).

Further analysis, integrated with Co-IP and LC–MS/MS, revealed that occludin interacted with three karyopherins, namely Importin α1, Exportin 1 and Exportin 2, which function as adapter proteins in nuclear protein import or export processes (Fig. 8K). As occludin contains a nuclear localization signal (NLS) (Fig. 8L), we confirmed significant interaction between occludin and Importin α1 in the ITGA3-HA group, whereas this interaction was absent in the N265A-HA group (Fig. 8M). In contrast to its predominant membrane localization in N265A-HA, occludin was primarily distributed in the nuclear region through interaction with Importin α1 in the ITGA3-HA group (Fig. 8N), suggesting that the sLeX-modified ITGA3 facilitates the nuclear translocation of occludin, possibly in an Importin α1-dependent manner.

During the process of metastasis, cancer cells detach from the primary site, enter the bloodstream, and eventually establish themselves in distant organs [37, 38]. One critical factor in this cascade is the elevation of vascular permeability, which helps in the dissemination of cancer cells and fosters their colonization at distant sites [39].

The expression of sLeX, a tetrasaccharide epitope displayed on circulating cells, has been found to be increased in various types of cancers and linked to cancer cell engagement with targeted endothelium through interaction with selectins [20, 40]. A recent study showed that engineering two sLeX peptides onto EVs significantly increased specificity towards activated colon endothelial cells [41]. In this study, we demonstrated that high level of sLeX can promote bladder tumor growth and invasion. Furthermore, we observed heightened sLeX levels on sEVs originating from invasive BLCA cells. It is well-established that sEVs released by tumor cells exert a profound influence on different stages of tumor metastasis. This includes their involvement in disrupting cell junctions, enhancing blood vessel permeability, and priming the microenvironment in distant organs to facilitate the colonization of cancer cells [10, 42, 43]. Consistently, our study observed that sEVs with high sLeX modification were efficiently endocytosed by HUVECs, consequently disrupting the tight junctions of endothelial cells and leading to increased vascular permeability.

Using antibody-based enrichment and MS analysis, we identified vesicular ITGA3 as a pivotal sLeX-bearing protein. Notably, ITGA3 expression was found to be markedly linked to the occurrence of intravesical recurrence in non-muscle-invasive BLCA [44]. Moreover, the presence of ITGA3 on sEVs was detected in exosomes derived from the urine of metastatic prostate cancer patients [45]. In line with these findings, our study indicated that ITGA3 is abundantly expressed in BLCA tissues and is significantly associated with patients' adverse prognosis. It is well-known that integrins are typical transmembrane glycoproteins [46]. Proper glycosylation is essential for their correct folding and trafficking, whereas aberrant glycosylation or its removal can impact the stability and degradation of integrins [47, 48]. Despite extensive research on the function of glycosylation in various integrins, the glycosylation status of ITGA3 has not been clearly defined. Our study revealed that ITGA3 undergoes substantial sLeX modification in BLCA, and conversely, sLeX modification serves to shield ITGA3 from lysosomal degradation. The mutation (A349S) on ITGA3 leads to a gain of glycosylation, impeding the heterodimerization of ITGA3 precursor with integrin β1 and diminishing the distribution of ITGA3 at the cell surface, further leading to severe kidney and lung defects [49]. Furthermore, our study pinpointed the sLeX modification site of ITGA3 at N265, which enhances its stability by obstructing the lysosomal degradation pathway.

Integrins play a crucial role in the cellular migration process, thereby influencing cancer progression and development from the primary site to distant metastasis [15]. Integrin α3β1 has the ability to interact with laminin, thereby inducing vascular permeability [50]. ITGA3 promotes vessel formation of glioblastoma-associated endothelial cells through enhancing calcium influx and micropinocytosis [51]. In our study, we demonstrated that sLeX-decorated ITGA3 on sEVs downregulates TJ protein expression, alters its subcellular localization, disrupts vascular endothelial barriers, and induces angiogenesis via the MAPK signaling pathway (Fig. 9). When ITGA3 was silenced in FUT7 overexpressed donor cells, sEVs decorated with sLeX but lacking ITGA3 exhibited attenuated tumor angiogenesis and restored vascular barriers, without compromising the efficiency of sEV uptake by HUVECs.

The TJ structure is crucial for maintaining the functional integrity of the endothelial barrier. Occludin, a key structural component of TJ filaments, undergoes hyperphosphorylation on Ser/Thr residues within the intact TJ structure [36], while the subcellular localization of occludin can be altered by the regulation of protein kinases and phosphatases [52]. The mutations at tyrosine residues (Y398D/Y402A) resulted in occludin predominately distributing within the intracellular compartment during TJ assembly process [36]. PKCη (a novel PKC isoform) can induce the distribution of occludin from the intracellular compartment to the intercellular junction by phosphorylating T403 and T404 residues [53]. Our findings indicate that sLeX-modified ITGA3 enhances the interaction between occludin and PP1A in HUVECs, thereby facilitating the dephosphorylation of occludin on Ser/Thr residues by PP1A. This sequential process subsequently promotes the translocation of occludin into the nucleus mediated by Importin α1, leading to the activation of the MAPK signaling pathway and disruption of the TJ structure between endothelial cells (Fig. 9).

In summary, our data characterized the high level of sLeX on sEVs in BLCA, and revealed the importance of sLeX modified ITGA3 on sEVs in regulating vascular endothelial barriers by altering phosphorylating status of occludin at Ser/Thr residues and its subcellular localization, thereby facilitating tumor metastasis. The study might offer a promising approach for combating metastasis in malignant bladder cancer.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 31971211, No. 82172828 and No. 32071274). Science Foundation for Distinguished Young Scholars of Shaanxi Province (No. 2021JC-39), the Natural Science Foundation of Shaanxi Province (No. 2021SF-294), Shaanxi Innovation Team Project (No. 2023-CX-TD-58) and the Youth Innovation Team of Shaanxi Universities.

Author contributions

FG conceived the study. FG and HF designed experiment and analysis. HF, WD and JG performed experiments. HF analyzed the data and wrote the original draft. LL provided clinical resources. XL and FG edited the manuscript in collaboration with HF. All authors have reviewed and approved the final version of the manuscript

Competing interest

The authors declare no competing interests.

Ethical approval

With the approval of the Ethics Committee of Northwest University, we obtained human plasma from bladder cancer patients and healthy volunteers in the First Affiliated Hospital of Xi'an Jiaotong University and the Shaanxi Provincial People's Hospital. All animal experiments are approved by the Institutional Animal Care and Use Committee of Northwest University.

Availability of data and material

The resource data supporting this paper are presented within the Supplementary Materials. The data that support the findings of this study are available from the authors upon reasonable request.

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SLeX decorated integrin α3 on sEVs promotes metastasis of bladder cancer via enhancing vascular permeability

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Journal Publication

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Abstract

Figures

Introduction

Materials and methods

Patient samples

Identification of proteins with sLeX

Tissue microarray/immunohistochemical analysis

Animal models

Statistical analysis

Results

Expression of FUT7 and sLeX was positively associated with BLCA metastasis

SLeX promoted the malignant progression of BLCA

BLCA derived-sEVs increased vascular permeability in vivo and in vitro

SLeX modified-vesicles were efficiently uptaken by HUVECs and destroyed vascular endothelial barriers

Vesicular ITGA3 was identified as a target protein modified by sLeX

ITGA3 promoted vascular permeability and angiogenesis

Silencing ITGA3 reversed sLeX induced tumorigenesis and metastasis

SLeX-modified ITGA3 induced occludin translocation into nuclear

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1