Andrographolide targets syndecan4 to impair its interaction with syntenin and inhibits the biogenesis of small extracellular vesicles

TL;DR

This study shows that Andrographolide, a natural compound, inhibits the production of tumor-promoting small extracellular vesicles by targeting Syndecan4.

Contribution

The study identifies Andrographolide as a novel compound that disrupts Syndecan4-Syntenin interaction and reduces extracellular vesicle biogenesis.

Findings

Syndecan4 undergoes constitutive shedding and is degraded via γ-secretase and proteasome pathways.

Andrographolide directly binds to Syndecan4 and blocks its interaction with Syntenin.

Andrographolide promotes lysosomal degradation of Syndecan4 and reduces small extracellular vesicle production.

Abstract

Syndecan4 (SDC4), a well-characterized plasma membrane glycoprotein that functions as an extracellular matrix receptor and growth factor co-receptor, is frequently overexpressed in tumors. Its accumulation is associated with increased generation of small extracellular vesicles (sEVs), promoting tumor development and metastasis. However, the underlying mechanism of SDC4 degradation remains poorly understood. This study reveals that SDC4 undergoes constitutive shedding, generating a transmembrane C-terminal fragment (CTF). This fragment is subsequently cleaved by γ-secretase, leading to rapid, likely proteasome-dependent degradation under basal conditions, thereby maintaining SDC4 homeostasis. During endocytic or stress conditions, SDC4-CTF is alternatively degraded via the endocytosis-lysosome pathway. Overexpression of Syntenin protects SDC4-CTF from endo-lysosomal degradation. To identify small molecules that directly modulate SDC4 stability, we employed a microscale thermophoresis screening approach and identified Andrographolide (AGO), a natural compound that directly binds to SDC4 and selectively blocks its interaction with Syntenin. Further studies have shown that AGO promotes SDC4-CTF lysosomal degradation and reduces the production of sEVs. In summary, our findings demonstrate that AGO blocks SDC4-Syntenin interaction and inhibits sEVs biogenesis, providing a new pharmacological ligand for targeting SDC4.

Figures

Full text

Syndecans (SDCs) play crucial roles in tumorigenesis, angiogenesis, and neural development, with strong evidence demonstrating their involvement in the development of various cancers, including breast, prostate, colorectal, and pancreatic carcinomas (1, 2, 3, 4). Notably, SDC1 is significantly overexpressed in pancreatic cancer tissues compared to normal counterparts, correlating with disease progression and poor prognosis (5). Recent studies demonstrate that KRAS upregulates SDC1 to drive macropinocytosis, thereby promoting pancreatic cancer cell growth (6). Our previous study suggests that SDC4, similar to SDC1, is essential for the development of pancreatic and colorectal cancers (7). The CRISPR-Cas9-mediated KO of SDC4 in PANC1 and HCT116 cells drastically impairs their clonogenic capacity and effectively suppresses subcutaneous xenograft tumor formation and growth. SDC4 was also identified as an attractive therapeutic target for hepatocellular carcinoma (8). Furthermore, SDC4 overexpression is well-documented in gastric cancer, particularly the intestinal subtype, and correlates with diminished patient survival (9). Mechanistic investigations identify SDC4 as a master regulator of invasion and migration in gastric cancer cells, driving metastatic progression.

SDCs proteins are essential for the biogenesis of small extracellular vesicles (sEVs), which are 30 to 150 nm secretory vesicles released by most cells in the body (10, 11, 12, 13). sEVs facilitate intercellular communication and are involved in various physiological and pathological processes (14). SDCs localize to atypical lipid domains and, together with the small intracellular PDZ scaffolding protein Syntenin, contribute to sEVs formation and cargo composition (10). The Syndecan-Syntenin-ALIX ternary complex regulates approximately 50% of secretory vesicles and plays a significant role in sEVs biogenesis. Gain- and loss-of-function studies show that these three components coordinately regulate the release of sEVs, as well as proteins like CD63 and heat shock protein 70 (12). Moreover, heparan sulfate-modified SDC4 is efficiently sorted into sEVs, and SDC4 in sEVs controls the distribution, uptake, and functional properties of gastric cancer-derived sEVs in recipient cells (9). Ablation of SDC4 disrupts the tropism of sEVs for common gastric cancer metastatic sites. Collectively, these findings establish SDC4 as a key regulator of sEVs biogenesis and function, presenting novel therapeutic opportunities targeting the SDC4-sEVs axis to impede cancer progression (15).

Despite the important role of SDC4 in tumors and sEVs, the mechanism underlying its protein degradation remains unclear. It has been reported that non-canonical Wnt signaling promotes the mono-ubiquitination of the SDC4 cytoplasmic domain, leading to proteasome-dependent degradation (16). Conversely, another study found that SDC4 mainly undergoes lysosomal degradation rather than proteasomal turnover in MCF-7 cells (17). These conflicting findings have created confusion regarding SDC4 degradation mechanisms. Our study shows that both pathways contribute to SDC4 degradation in colorectal cancer cells. SDC4 is constitutively shed to produce a transmembrane (TM) C-terminal fragment (CTF), which is then cleaved by γ-secretase, leading to rapid, probably proteasome-dependent degradation. Overexpression of Syntenin stabilizes SDC4-CTF against endocytosis-lysosome degradation. We also identified Andrographolide (AGO), the main active component of Andrographis paniculata, which binds SDC4, blocks its interaction with Syntenin, and reduces SDC4 levels, especially CTF, by promoting lysosomal degradation in a cell-type-dependent manner. Additionally, AGO decreases SDC4-CTF in sEVs and inhibits their formation and uptake.

Results

MG132 inhibits the degradation of SDC4-CTF

The heparan sulfate-bearing ectodomain of SDC4 undergoes ligand-activated or stress-induced proteolytic cleavage and shedding (18, 19, 20). However, the fate of the residual membrane-associated CTF is not well studied. To assess whether proteasome is involved in the degradation of SDC4-CTF, we utilized the proteasome inhibitor MG132. Our findings indicate that MG132 significantly increases the protein levels of both SDC1-CTF and SDC4-CTF (Fig. 1, A and B), but not the full-length (FL) SDC1 and SDC4 proteins (Fig. 1C), in a concentration- and time-dependent manner in HCT116 cells. Furthermore, overexpression of C-terminally GFP-tagged SDC1 and SDC4 in HCT116 cells revealed a significantly increased in fluorescence intensity just 4 hours after treatment with 10 μM MG132 (Fig. 1, D and E). These results demonstrate that MG132 significantly inhibits the degradation of SDC4-CTF. This was validated across various colorectal cancer cell lines (Fig. 1F).Figure 1**MG132 inhibits the degradation of SDC4-CTF.**A and B, Western blot of HCT116 cells treated with 0.5, 1, 2.5, or 5 μM MG132 for 12 h, or with 2.5 μM MG132 for 0 to 12 h. C, Western blot of full-length SDC1 and SDC4 in HCT116 cells treated with MG132 (0.5–5 μM, 12 h). D and E, immunofluorescence detection and quantification of HCT116 cells transfected with SDC1-GFP or SDC4-GFP, with/without 5 μM MG132 (4 h; n = 6). F, Western blot of multiple colorectal cancer cell lines treated with 10 μM MG132 (12 h). G, Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells treated with 0.1, 0.5, or 1 μM proteasome inhibitors (Carfilzomib, Ixazomib, Bortezomib) for 24 h. H, Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells treated with PD150606 (2, 5, 10 μM) for 12 h. I, quantification of DQ-BSA fluorescence with MG132 or Earle’s balanced salt solution (EBSS) treatment (n = 4). J, quantification of lysosomal activity using LysoTracker with MG132 or EBSS treatment (n = 4). K, Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells with siRNA knockdown of proteasome subunits PSMD14, PSMD2, USP14, PSMB5, PSMA6. Data: mean ± SD (≥3 experiments). Statistics: unpaired two-tailed t test (∗∗∗p < 0.0005, ∗∗∗∗p < 0.0001). SDC4, syndecan4; CTF, C-terminal transmembrane (TM) fragment; EBSS, Earle’s balanced salt solution.

Unexpectedly, other three proteasome inhibitors, Carfilzomib, Ixazomib, and Bortezomib, failed to stabilize SDC1-CTF or SDC4-CTF (Fig. 1G). This may be due to their highly specific inhibition on proteasome, whereas MG132 inhibits multiple active sites (21). We also noticed that MG132 is a potent inhibitor of broad-spectrum proteases, including calpains and cathepsins, which are involved in lysosomal activity (22, 23, 24). To exclude non-specific effects, we tested the calpain inhibitor PD150606 and confirmed it does not stabilize SDC1-CTF or SDC4-CTF, indicating calpain is not involved in their degradation (Fig. 1H). To further rule out impact of MG132 on lysosome, we used Lysotracker and DQ-BSA (a BSA derivative heavily labeled with dyes, self-quenches but emits bright fluorescence when cleaved by proteases) to evaluate lysosomal activity. As expected, MG132 did not inhibit lysosomal activity but slightly increased the endocytosis of DQ-BSA (Fig. 1, I and J), suggesting MG132 stabilizes SDC4-CTF primarily through the proteasome pathway. This was further supported by knockdown of key proteasome subunits, which significantly stabilizes SDC1-CTF and moderately increases SDC4-CTF (Fig. 1K).

SDC4-CTF is further cleaved by γ-secretase to release the cytoplasmic fragment

SDC4-CTF retains the TM region, and fluorescence microscopy of C-terminally GFP-tagged SDC4-CTF (Fig. 2B) shows that its localization similar to SDC4-FL, mainly observed on the cell membrane (Fig. 2A). This raises the question of how the proteasome degrades membrane proteins. Literature review revealed that γ-secretase plays a crucial role in degradation of single-pass TM proteins (25, 26, 27). The endogenous product generated by γ-secretase cleavage, a C-terminal fragment (cCTF, similar in molecular weight but lacking a TM region) is indistinguishable from SDC4-CTF by conventional Western blotting. Therefore, we overexpressed exogenous SDC4-CTF-GFP in HCT116 cells to determine whether it could be further cleaved to produce a lower-molecular-weight band. Western blot confirmed that SDC4-CTF-GFP indeed can be cleaved into a lower-molecular-weight fragment, SDC4-cCTF-GFP, which is significantly stabilized by MG132 (Fig. 2C). Notably, overexpressed SDC4-GFP appeared as two distinct bands, indicating that SDC4 undergoes constitutive shedding. The remaining CTF is further cleaved by γ-secretase to release cCTF that is more sensitive to MG132, similar to SDC1 (Fig. 2C).Figure 2**SDC4-CTF is further cleaved by γ-secretase to release the cytoplasmic fragment.**A, confocal microscopy of HCT116 cells transfected with SDC4-GFP or SDC4-CTF-GFP. B, schematic of SDC4-GFP deletion mutants, SNP for Signal peptide, ED for ectodomain, TM for TM motif, C1 and C2 for the constant regions, and V for variable region. C, Western blot of HCT116 cells transfected with SDC1-GFP, SDC4-GFP, or SDC4-CTF-GFP, with/without 5 μM MG132 (4 h). D, sequence alignment of Syndecan family TM C-terminal fragments. The alignment was performed using Clustal W, and analysis was done with ESPript 3.0. E, Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells with presenilin-1 knockdown (siRNA, 72 h). F, Western blot of HCT116 and SW480 cells treated with 10 μM DAPT, 10 μM MG132, or 2.5 μM GM6001 (12 h). G, schematic of SDC4-CTF degradation via lysosome or proteasome pathways. Data: mean ± SD (≥3 experiments). Statistics: unpaired two-tailed t test (∗∗p < 0.001, ∗∗∗p < 0.0005, ∗∗∗∗p < 0.0001). SDC4, Syndecan4; CTF, C-terminal transmembrane fragment.

As reported, γ-secretase recognizes and cleaves membrane proteins containing GxxxG motifs in the TM region, releasing their intracellular domains from the cell membrane for proteasomal degradation (28, 29, 30). We then compared the amino acid sequences of SDCs and found that their TM regions all contain the GGxxG motif, indicating they are potential substrates for γ-secretase (Fig. 2D). We next performed siRNA-mediated knockdown of presenilin-1, the catalytic subunit of γ-secretase. This approach consistently demonstrated that SDC4-CTF cleavage is γ-secretase-dependent (Fig. 2E). Additionally, the γ-secretase-specific inhibitor DAPT, like MG132, significantly stabilizes SDC1-CTF and SDC4-CTF in HCT116 and SW480 cells (Fig. 2F). These results strongly suggest γ-secretase involvement in SDC4-CTF proteolytic degradation. Interestingly, co-treatment with the matrix metalloproteinase inhibitor GM6001 significantly decreased the stabilizing effects of both DAPT and MG132 on SDC1-CTF and SDC4-CTF (Fig. 2F), demonstrating that ectodomain shedding is the initial and crucial step, followed by γ-secretase cleavage and proteasome degradation. Notably, SDC1-CTF was more responsive to DAPT and MG132 than SDC4-CTF, suggesting SDC1-CTF is less stable and more rapidly cleaved by γ-secretase and proteasome. Consistent with this, SDC1-CTF was more difficult to detect by Western blot than SDC4-CTF. Collectively, these data indicate that shedding-γ-secretase-proteasome degradation also contributes to SDC4 homeostasis (degradation model shown in Fig. 2G).

Syntenin stabilizes SDC4-CTF against endocytic-lysosomal degradation

We further examined the impacts of autophagy and lysosomal inhibitors on SDC1-CTF and SDC4-CTF. As previously documented (31, 32, 33), the lysosomal inhibitor Bafilomycin A1 (BAF1) significantly elevated the protein levels of both SDC1-CTF and SDC4-CTF (Fig. 3A). The autophagy inhibitor LY294002 stabilized SDC1-CTF, while the autophagy inducer Rapamycin had the opposite effect; however, neither influenced SDC4-CTF (Fig. 3A), indicating that SDC4-CTF might not degrade via the autophagy-lysosome pathway. In contrast, starvation treatment with Earle’s balanced salt solution induced rapid degradation of SDC4-CTF within 0.5 h without affecting SDC1-CTF (Fig. 2B), suggesting SDC4-CTF is predominantly degraded via starvation-induced endocytosis-lysosomal pathways. Additionally, the Syntenin protein, which directly interacts with SDCs through its PDZ domain (12), was also degraded via this endocytosis-lysosome pathway (Fig. 3B), indicating Syntenin might play an essential role in SDC4 degradation.Figure 3**Syntenin stabilizes SDC4-CTF against endocytic-lysosomal degradation.**A, Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells treated with 25 μM LY294002, 250 nM Rapamycin, or 250 nM Bafilomycin A1. B, Western blot of HCT116 cells starved in EBSS for indicated durations. C and D, Western blot and quantification of SDC1-CTF and SDC4-CTF in HCT116 cells overexpressing Flag-tagged Syntenin (n = 3). E, schematic and sequences of SDC4^WT^ and SDC4^ΔC2^, with confocal images of indicated plasmids in 293T cells. F, quantification of fluorescence co-localization from (E) (n = 6). G and H, Western blot and quantification of SDC4-CTF and Syntenin in 293T cells co-transfected with SDC4 and GFP-tagged Syntenin (n = 3). I, Western blot of SDC1-CTF, SDC4-CTF, and Syntenin in multiple cancer cell lines. J, correlation analysis between SDC4-CTF and Syntenin expression from (I). Data: mean ± SD (≥3 experiments). Statistics: unpaired two-tailed t test (∗∗p < 0.001, ∗∗∗p < 0.0005, ns). SDC4, Syndecan4; CTF, C-terminal transmembrane fragment; EBSS, Earle’s balanced salt solution.

To explore whether Syntenin binding stabilizes SDC4-CTF, we overexpressed Flag-tagged Syntenin in HCT116 cells. Western blot analysis revealed a slight increase in SDC4-CTF levels in Flag-Syntenin overexpressing cells (Fig. 3, C and D). To confirm that this effect stems from direct interaction between the two proteins, we overexpressed WT or an SDC4 mutant with a deleted PDZ-binding motif (ΔC2) alongside GFP-tagged Syntenin in 293T cells (Fig. 3E). Confocal microscopy showed that Syntenin was markedly recruited to the plasma membrane by SDC4^WT^, but remained mostly cytosolic when SDC4^ΔC2^ was expressed (Fig. 3, E and F). Additionally, the fluorescence intensity of SDC4 was notably stronger in WT cells (Fig. 3E). Western blotting yielded consistent results that SDC4-CTF was significantly higher in WT cells compared to ΔC2 mutant (Fig. 3, G and H). Finally, we evaluated SDC4-CTF and Syntenin expression levels across several cancer cell lines. The results showed that cell lines with evaluated SDC4-CTF (SW480, HCT15, DLD1, A375, and HCC827) also exhibited high Syntenin protein levels. Spearman correlation analysis confirmed a similar expression pattern between SDC4-CTF and Syntenin (Fig. 3J). These findings collectively demonstrate that Syntenin stabilizes SDC4-CTF against endocytic-lysosomal degradation.

AGO directly binds to SDC4 and specifically inhibits its interaction with syntenin

SDC4 is an intrinsically disordered protein, and traditional structure-based design methods are unsuitable for identifying small-molecule binders (34, 35, 36). To identify compounds that directly modulate SDC4 stability, we employed a previously developed microscale thermophoresis (MST) assay capable of assessing direct interactions between small molecules and target proteins in biological fluids (37). Using this approach, we previously identified an FDA-approved drug, Eltrombopag that directly binds to SDC4 (7). Here, we identified another compound, Andrographolide (AGO), a diterpenoid lactone, which also binds to SDC4 (Fig. 4A). Direct binding of AGO to SDC4 was validated using purified glutathione S-transferase (GST)-tagged recombinant SDC4 proteins. Dissociation constants (Kd) for AGO binding were 47 nM in cell lysate and 810 nM with purified protein (Fig. 4, B and C). The ∼20-fold difference in Kd value may be attributed to the complexity of cell lysate, SDC4 modifications, and formation of complexes (38). AGO also showed strong affinity for SDC1, with a Kd of 178.6 nM (Fig. 4E). Further analysis indicated that TM motif of SDC4 and SDC1 is crucial for AGO engagement (Fig. 4, D and E). Drug affinity responsive target stability assay demonstrated that AGO stabilizes SDC4, making it less susceptible to proteolysis further supports our conclusion (Fig. 4, F and G).Figure 4**AGO binds to SDC4 and disrupts SDC4-Syntenin interaction.**A, chemical structure of AGO. B, microscale thermophoresis (MST) assays of AGO with GFP-SDC4 or GFP control in 293T lysate (n = 3). C, MST assays of AGO with recombinant glutathione S-transferase (GST)-SDC4 (n = 3). D and E, MST assays of AGO with GFP-tagged SDC4 or SDC1 and their TM deletion mutants (n = 3 each). F and G, Western blot and quantification of AGO drug affinity responsive target stability experiments (n = 3). H and I, immunoprecipitation (IP) and quantification of SDC4 binding to Syntenin in SW480 cells treated with 20 μM AGO (24 h; n = 3). J, IP of Flag-Syntenin and GFP-SDC4 in 293T cells treated with 20 μM AGO (24 h). K, GST pull-down of Syntenin from 293T lysate with/without 20 μM AGO, using GST or GST-SDC4. L and M, poximity ligation assays and quantification of SDC4-Syntenin interaction in SW480 cells treated with 20 μM AGO (24 h; n = 8). O and P, confocal images and quantification of SDC4/GFP-Syntenin co-localization in 293T cells treated with 20 μM AGO (24 h; n = 8). Q, IP of SDC4 and detection of GFP-Syntenin/endogenous Syntenin in 293T cells treated with 20 μM AGO (24 h). Data: mean ± SD (≥3 experiments). Statistics: unpaired two-tailed t test (∗∗p < 0.001, ∗∗∗p < 0.0005, ns). AGO, Andrographolide; GST, glutathione S-transferase; SDC4, Syndecan4; MST, microscale thermophoresis; TM, transmembrane; IP, immunoprecipitation.

We then examined whether AGO binding affects SDC4’s interaction with its partners. Anti-SDC4 immunoprecipitation (IP) in SW480 cells with high SDC4 expression was performed to assess four known interactors: β-catenin, Syntenin, Rab5, and α-tubulin (39, 40, 41). Notably, only the interaction with Syntenin was significantly reduced by AGO (Fig. 4, H and I). Reverse IP also showed that AGO significantly reduced Flag-immunoprecipitated SDC4 levels in 293T cells co-overexpressing Flag-tagged Syntenin and SDC4-GFP (Fig. 4J). GST pull-down assays with purified GST-tagged SDC4 confirmed that AGO markedly reduces SDC4-Syntenin interaction (Fig. 4K). Proximity ligation assays also supported this finding, showing that AGO disrupts the interaction between SDC4 and Syntenin (Fig. 4, L and M). To visualize this effect more clearly, we performed confocal microscopy on 293T cells co-expressing SDC4 and GFP-tagged Syntenin with AGO treatment. The results suggest that AGO disrupts their co-localization, further supported by IP data (Fig. 4, O–Q).

AGO promotes SDC4 endocytosis-lysosome degradation in a cell type-dependent manner

SDC4 is highly expressed in tumors and promotes metastasis (38). We were inspired to examine the effect of AGO on SDC4 turnover. As expected, treatment with AGO significantly decreased the protein levels of SDC1-CTF and SDC4-CTF in a concentration- and time-dependent manner in SW480 cells, but did not affect the abundance of SDC1-FL and SDC4-FL (Fig. 5, A and B). While immunofluorescence and flow cytometry analyses showed that AGO decreased cell-surface SDC4 levels in SW480 cells (Fig. 5, C–E). This paradoxical result may reflect heterogeneity among SDC4-FL forms due to glycosylation and oligomerization (42). Interestingly, AGO only slightly decreased SDC1-CTF and SDC4-CTF in HCT116 cells (Fig. 5F). To determine if AGO’s effect is cell type-dependent, we examined a panel of colorectal cancer cell lines. Upon AGO treatment, compared with HCT116, SW620, and DLD1, SDC4-CTF in HCT15, SW480, and RKO cells was significantly down-regulated (Fig. 5G), confirming cell type-dependent effects of AGO.Figure 5**AGO promotes lysosomal degradation of SDC4-CTF.**A, Western blot of SDC1/4 CTF and full-length in SW480 cells treated with AGO (indicated doses, 24 h; n = 3). B, Western blot in SW480 cells treated with 20 μM AGO for 0 to 24 h. C and D, immunofluorescence detection and quantification of SDC4 in SW480 cells treated with 20 μM AGO (24 h; n = 6). E, surface SDC4 measured by FACS using FITC-anti-SDC4. F, Western blot of SDC1/4 CTF and full-length in HCT116 cells treated with AGO (indicated doses, 24 h). G, Western blot of SDC4-CTF in multiple colorectal cancer cells treated with 20 μM AGO (24 h). H, Western blot of SDC1-CTF and SDC4-CTF in SW480 cells treated with cycloheximide (50 μg/ml) along with/without 20 μM AGO for indicated times. I, RT-qPCR of indicated genes in SW480 cells treated with 20 μM AGO (24 h; n = 4). J, Western blot of SDC1-CTF and SDC4-CTF in SW480 cells treated with 20 μM AGO, 2.5 μM GM6001, 10 μM TMI-1, or combinations (24 h). K, Western blot in SW480 cells treated with 20 μM AGO, 5 μM BAY11 to 7082, 10 nM PMA, 1 μg/ml LPS, or 20 μg/ml TNFα (24 h). L and M, live-cell imaging and analysis of SDC4-GFP intensity in 293T cells co-expressing DsRed-Rab5A and SDC4-GFP, with 20 μM AGO treatment (n = 4). O, Western blot of lysosome markers and SDC1/4-CTF in SW480 cells treated with 20 μM AGO, 250 nM BAF1, or combination (24 h). Data: mean ± SD (≥3 experiments). Statistics: unpaired two-tailed t test (∗p < 0.05, ∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns). AGO, andrographolide; SDC4, Syndecan4; CTF, C-terminal transmembrane fragment; FACS, fluorescence-activated cell sorting; BAF1, Bafilomycin A1.

To assess whether AGO promotes the degradation of SDC1-CTF and SDC4-CTF, we used cycloheximide (CHX) chase assays. In the presence of CHX, AGO caused further reductions of SDC1-CTF at 12 h but did not enhance SDC4-CTF decrease (Fig. 5H), possibly because CHX is unsuitable for proteins with slow degradation rates (43). RT-PCR analysis showed AGO slightly decreased the mRNA levels of SDC1 and SDC4 while significantly downregulated MMP2 and MMP9 mRNA levels as previously reported (Fig. 5I) (44). To test if the reduction of SDC4-CTF by AGO results from inhibited shedding, SW480 cells were treated with matrix metalloproteinase inhibitors GM6001 and TMI-1 along with AGO. Neither inhibitor blocked AGO's effect (Fig. 5J), indicating AGO decreased SDC4-CTF was not due to shedding inhibition. Since SDC4 is an oncogenic NF-κB target gene induced by TNFα in tumor cells (45), and AGO can covalently modify a reduced cysteine within the oligonucleotide-binding pocket of p50, leading to NF-κB inhibition (46), we tested if AGO suppresses SDC4 transcription via the NF-κB pathway. Another NF-κB specific inhibitor, BAY11-7082 (BAY), was used to mimic AGO's activity. Interestingly, BAY did not reduce SDC4-CTF levels, whereas activators TNFα and PMA significantly decreased SDC4-CTF and increased SDC1-CTF (Fig. 5K). This excludes the possibility that AGO inhibits SDC4 transcription through NF-κB inhibition and other mechanisms may be involved.

Rab5 is essential for SDC4 endocytosis-lysosome degradation, and its overexpression drives SDC4 into early endosomes destined for lysosomal degradation (12). To investigate if AGO promotes this process, we co-overexpressed DsRed-Rab5A and SDC4-GFP in 293T cells treated with 20 μM AGO. Live cell imaging was used to track the real-time dynamics of SDC4-GFP over 21 h, with data collected every 3 h. The results showed that AGO did not enhance SDC4-Rab5 co-localization while reduce SDC4-GFP mean intensity during AGO incubation (Fig. 5, L and M), suggesting AGO might enhance the endocytosis-lysosome degradation of SDC4. Moreover, lysosomal markers LAMP2, p62, and LC3 were significantly increased by AGO treatment, and co-treatment with BAF1 notably attenuated AGO's effect on SDC1-CTF and SDC4-CT (Fig. 5O). Collectively, these data demonstrate that AGO promotes lysosomal degradation of SDC4 in a cell type-dependent manner, although detailed mechanism needs further investigation.

AGO inhibits the generation and function of sEVs

As previously established, SDC4 and Syntenin play a crucial role in sEVs release (12). Our data above show that AGO impairs SDC4-Syntenin interaction and reduces SDC4-CTF levels in a cell-type-dependent manner. In light of this, we investigated the potential impact of AGO on sEVs composition and production. sEVs from SW480 cells treated with or without AGO were collected and analyzed by Western blotting. The results showed that AGO significantly reduced the levels of SDC1-CTF and SDC4-CTF in sEVs, with a slight downregulation of Syntenin (Fig. 6A). Nanoparticle tracking analysis revealed AGO significantly decreased the total number of secreted particles (Fig. 6B). The sEVs secreted by AGO-treated cells exhibited a smaller size (Fig. 6C). Additionally, transmission electron microscopy demonstrated that AGO notably inhibited multivesicular bodies (the main source of sEVs) formation in SW480 cells (Fig. 6, D and E). The PKH67 fluorescently labeled sEVs uptake experiment showed that sEVs from AGO-treated cells were captured less efficiently by HeLa cells (Fig. 6, F and G). These findings suggest that AGO inhibits the generation and function of sEVs at the cellular level, warranting further investigation in animal models.Figure 6**AGO inhibits the production and function of sEVs.**A, Western blot of sEVs markers in cell lysate and sEVs from SW480 cells treated with 20 μM AGO for 40 h. B and C, concentration and size distribution (nanoparticle tracking analysis) of sEVs from SW480 cells with/without 20 μM AGO (n = 3). D and E, transmission electron microscopy ultrastructural analysis and quantification of multivesicular body compartments in SW480 cells with/without 20 μM AGO (24 h; n = 4). F and G, uptake of PKH67-labeled SEVs (green) analyzed by immunofluorescence and quantified (n = 16). HeLa cells were incubated with sEVs from SW480 cells with/without 20 μM AGO treatment (2 h). Data: mean ± SD (≥3 experiments). Statistics: unpaired two-tailed t test (∗∗p < 0.001, ∗∗∗p < 0.0005, ∗∗∗∗p < 0.0001). AGO, andrographolide; sEVs, small extracellular vesicles.

Discussion

Abnormal expression of heparan sulfate proteoglycans is observed in various tumor models (47). Notably, SDC4 is upregulated in glioma, melanoma, osteosarcoma, papillary thyroid carcinoma, as well as breast, liver, kidney, and bladder carcinomas (38). Our previous studies highlighted the critical role of SDC4 in the progression of pancreatic and colorectal cancers (7). Knocking out SDC4 in PANC1 and HCT116 cells decreases clonogenic capacity and reduces subcutaneous xenograft tumor formation and growth. This study elucidates SDC4 degradation mechanism, showing degradation occurs via both proteasome and lysosome pathways. At steady state, cellular SDC4 mainly exists in two forms: SDC4-FL (25–35 kDa) and SDC4-CTF (∼15 kDa), the latter representing membrane remnants after proteolytic shedding. Shedding is essential for maintaining the homeostasis of single-pass TM proteins (48). We found that SDC1 and SDC4 in colorectal cells undergo continuous shedding, leading to the formation of CTF. Prior studies indicate that the SDC1-CTF can mimic the functions of SDC1-FL, promoting proliferation, wound healing, and invasion in cells lacking endogenous SDC1 (49). Moreover, expressing this fragment enhances proliferation of BT-549 human breast cancer cells even when endogenous SDC1 is present (50). The conserved cytosolic domains of SDCs are vital for membrane signaling (41). However, the fate of the SDC4-CTF that remains in the membrane after shedding is poorly studied.

Here, we demonstrate that MG132 significantly increases the protein levels of both SDC1-CTF and SDC4-CTF in a concentration- and time-dependent manner (Fig. 1, A and B). While our data show that knocking down proteasome subunits or treating with the broad-spectrum inhibitor MG132 effectively stabilizes the SDC4-CTF, the absence of a similar effect when using multiple highly selective proteasome inhibitors (Carfilzomib, Ixazomib, and Bortezomib) creates ambiguity. This discrepancy suggests two possible explanations: (i) an indirect mechanism, where stabilization results from inhibiting other non-proteasomal proteases (such as certain calpains or cathepsins) that are known to be affected by MG132 or proteasome subunit knockdown, rather than a direct effect on the proteasome; or (ii) a specific but direct mechanism involving a proteasome variant or associated complex that is resistant to the selective inhibitors used. As a result, the specific machinery responsible for SDC4-CTF degradation has not yet been definitively identified.

Further analysis revealed that the CTF undergoes additional intra-membrane proteolysis by γ-secretase, releasing cCTF (Fig. 2, C–G). Consistent with our results, previous research has shown that SDC1 and SDC3 undergo regulated intramembrane proteolysis sensitive to γ-secretase inhibition (49, 51). The highly conserved GGXXG motif of SDCs plays a key role in γ-secretase cleavage (Fig. 2D) (51). Given the loose sequence specificity of γ-secretase and the significant homology within the TM and cytosolic domains among SDCs, we expect all SDC-derived CTFs to be further cleaved by γ-secretase. However, sensitivity to cleavage varied among SDCs, likely influenced by their polymerized state and modifications. Further research is needed to understand how γ-secretase is recruited to cleave SDCs-CTF. We also found that SDC4-CTF is more stable and less sensitive to γ-secretase cleavage than SDC1-CTF, as indicated by its lower sensitivity to DAPT and MG132. Consequently, SDC4-CTF was significantly degraded by micro-autophagy caused by starvation within 4 hours, while SDC1-CTF remained stable (Fig. 3B). Notably, Syntenin was co-degraded with SDC4-CTF. Additionally, impaired interaction between Syntenin and SDC4 significantly promoted SDC4-CTF endocytic-lysosomal degradation (Fig. 3, E–H). This was in line with the previous study that Syntenin controls ARF6-mediated recycling of SDCs through endosomal compartments, and its loss directs internalized SDCs toward lysosomes for degradation (52).

We previously developed a single-point microscale thermophoresis screening assay to identify small molecules targeting SDC4 (7). Using this method, we identified AGO, which binds directly to SDC4 and significantly inhibit its interaction with Syntenin (Fig. 4). Moreover, AGO notably reduces SDC4, especially the CTF form (Fig. 5, A–E). Mechanistic studies showed that AGO does not inhibit SDC4 shedding but promotes its lysosome degradation in a cell-type-dependent manner. While AGO also slightly decreased the SDC1 and SDC4 mRNA levels (Fig. 5I), transcription inhibition alone cannot fully explain AGO’s effects. To further determine whether AGO suppresses SDC4 transcription by inhibiting NF-κB pathway, we used another NF-κB specific inhibitor, BAY11-7082, to replicate AGO's activity. Unexpectedly, BAY11-7082 did not decrease SDC4-CTF levels, indicating that NF-κB-mediated transcriptional suppression is not the main mechanism (Fig. 5K). This finding differs from previous studies (45, 53), which may be due to cell-type-dependent effects and suggests other mechanisms may exist; consistent with AGO’s known multi-targeting nature (54).

In conclusion, our study demonstrates SDC4 can be degraded through two distinct pathways: proteasome and lysosome. We also identified AGO as a compound that directly binds to SDC4, disrupts its interaction with Syntenin, promotes its lysosomal degradation, and impairs the production and molecular composition of sEVs. This work systematically investigates the degradation mechanism of SDC4 and introduces a new pharmacological inhibitor targeting the Syndecan-Syntenin-sEVs pathway, with significant potential for sEVs research and cancer treatment. However, whether AGO's inhibition of sEVs depends solely on SDC4 requires confirmation at the cellular and animal levels via KO or knockdown experiments, and the detailed mechanism of AGO-induced SDC4 decrease warrants further investigation.

Experimental procedures

Chemicals and reagents

Andrographolide (HY-N0191), MG132 (HY-13259), Bortezomib (HY-10227), Carfilzomib (HY-10455), Ixazomib (HY-10453), DAPT (HY-13027), GM6001 (HY-15768), TMI-1 (HY-101448), LY294002 (HY-10108), Rapamycin (HY-10219), Bafilomycin A1 (HY-100558), cycloheximide (HY-12320), BAY 11 to 7082 (HY-13453), PMA (HY-18739), LPS (HY-D1056), and TNFα (HY-P70426) were purchased from MedChemExpress. Pronase (10165921001) was purchased from Sigma. Earle’s balanced salt solution buffer (24010043) was purchased from Thermo Fisher Scientific. DQ-BSA (D-12050SB) and Lysotracker (A1163847) were purchased from ShareBio. SDC4 rabbit polyclonal antibody (NB110–41551) was purchased from Novus for SDC4-CTF detection. SDC1 (12,922) for SDC1-CTF detection, PSMB5 (12,919), Phospho-p65 (3033), Rab5 (3547), and GAPDH (5174) antibodies were obtained from Cell Signaling Technology. Antibodies against LC3 (14600-1-AP), LAMP2 (66301-1-Ig), p62 (18420-1-AP), Syntenin (22399-1-AP), CD63 (25682-1-AP), TSG101 (28283-1-AP), CD9 (20597-1-AP), α-tubulin (11,224–1-AP), PSMA6 (67695-1-Ig), PSMED2 (14748-1-AP), USP14 (67746-1-Ig), and SDC1 (10593-1-AP) for SDC1-FL detection were obtained from Proteintech. β-catenin (ab16051) and PSMD14 (ab109123) antibodies were obtained from Abcam. SDC4 (sc-12766) for SDC4-FL detection, immunoprecipitation, and immunofluorescence study, and Ubiquitin (sc-8017) antibodies were obtained from SantaCruz. Flag antibody (F1804) was acquired from Sigma. Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L) (111-035-003) and anti-Mouse IgG (H+L) (115-035-003) were obtained from The Jackson Laboratory. Goat anti-Mouse IgG (H+L) Alexa Fluor 488 and Goat anti-Rabbit IgG (H+L) Alexa Fluor 555 were purchased from Invitrogen.

Cell lines, cell culture, cell transfection

SW480, HCT116, DLD1, and RKO cells were obtained from the American Type Culture Collection. HEK293T, HT29, LS174T, SW620, HCC827, HeLa, and A375 cells were purchased from the National Collection of Authenticated Cell Cultures. RPMI1640 medium for HCT116, SW480, SW60, HT29, and HCC827 cells, DMEM medium for HEK293T, A375, LS174T, and RKO cells, and MEM medium for HeLa cells were purchased from Gibco. All cultures were supplemented with 10% FBS (Gemini) and penicillin-streptomycin (100 U/ml and 100 μg/ml, respectively; Gibco) and grown at 37 °C in a 5% CO2 humidified chamber.

Plasmids construction

SDC1, SDC4, and SDC4-CTF C-terminal tagged-GFP plasmids were synthesized by Genscript Biotechnology Inc., based on a mEmerald-N1 vector. The pCDNA3.1-SDC4-IRES-GFP-Syntenin construct was also synthesized by Genscript Biotechnology Inc. The pCDNA3.1-SDC4-ΔC2-IRES-GFP-Syntenin mutant was created using the Mut Express II Fast Mutagenesis Kit V2 (C2140–01, Vazyme Biotech). Briefly, the WT coding sequence was amplified with PCR primers containing the designed mutations. PCR products were incubated with Exnase II for homologous recombination, and then transformed into competent cells for clone selection. The mutant primers used in this study are shown in Table S1. Small interfering RNA (siRNA) oligonucleotides targeting indicated proteins were synthesized by GenePharma and the siRNA sequences are shown in Table S2. These oligos were transfected into cells at 100 nM using Lipofectamine 3000 (Invitrogen), and knockdown was confirmed at the protein level.

Western blot analysis

Cells were lysed with RIPA buffer (9806, CST) containing protease inhibitors (A32953, Thermo Fisher Scientific) at 4 °C. Protein concentrations were measured using a BCA protein assay kit (ZJ102, Epizyme Biotech). Proteins (10–30 μg per lane) were separated by SDS-PAGE and transferred onto PVDF membranes (P0807, Millipore). The membranes were blocked with 5% skim milk for 1 h and then incubated overnight with primary antibodies at 4 °C. Subsequently, the membranes were incubated with HRP-conjugated secondary antibody (1:10,000, 7074S, CST) for 1 h. Protein bands were visualized using enhanced chemiluminescence detection reagent (P10300, NCM Biotech). Signals were captured using the ChemiDoc MP Imaging System (Bio-Rad). Band intensities were quantified with ImageJ software (https://imagej.net/ij/index.html).

Immunofluorescence

Cells were seeded onto 12 mm glass coverslips in 24-well plates. On the second day, the cells were transfected with the indicated plasmid or treated with specified ligands. Then, cells were washed with PBS, fixed in 4% paraformaldehyde at room temperature for 15 min, with or without permeabilization using 0.3% Triton X-100 for 15 min, blocked with 5% BSA for another 15 min, and washed with PBS between each step. After blocking, cells were incubated with primary antibodies in 0.5% BSA/PBS overnight at 4 °C. Subsequently, Alexa Fluor 488 or Alexa Fluor 555-labeled secondary antibodies (A-11034, Invitrogen) were applied for 45 min, followed by DAPI staining. Images were acquired using Leica Application Suite X software (https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/) on a Leica Stellaris5 confocal microscope. For live cell imaging, 293T cells were co-transfected with DsRed-Rab5A and SDC4-GFP plasmids for 48 h. Subsequently, upon addition of 20 μM AGO, the cells were transferred to the live-cell imaging chamber to monitor SDC4-GFP dynamics for 21 h. Images were acquired at 3-h intervals using a Leica Stellaris five confocal microscope.

Microscale thermophoresis

According to a previous study (37), ten million HEK293T cells overexpressing EGFP alone or EGFP-tagged SDC4 were lysed in 0.5 ml RIPA buffer (9806, CST) containing protease inhibitors (36,978, Thermo Fisher Scientific). Cell lysates were diluted in PBS buffer to a final concentration suitable for detecting EGFP fluorescence signals on the Monolith NT.115 instrument (NanoTemper Technologies). AGO was serially diluted, and 10 μl of protein was mixed with 10 μl of compound at the specified concentration. Fluorescently labeled proteins were loaded into capillaries, and binding affinity (Kd) was determined using the Monolith NT.115 system. The purified proteins were labeled using the Red-NHS kit following the manufacturer’s protocol (MO-L011, NanoTemper Technologies). The mixture solutions were then loaded into NT.115 standard-coated or premium-coated capillaries (NanoTemper Technologies). Binding affinity was analyzed with the software supplied with the instrument.

Recombinant protein purification

The cDNA encoding human SDC4 (P31431) was synthesized (Genscript) and cloned into pGEX-6P-1 to enable N-terminal GST-tagging of the SDC4 protein. The construct was transformed into the expression host E. coli strain BL21 (DE3), and the cells were grown in YEP medium at 37 °C until the OD_600_ of 0.6. The cells were then induced by adding 0.1 mM IPTG to the culture and grown at 18 °C overnight. Cells were harvested by centrifugation. The cell pellet was resuspended in a buffer containing 20 mM Tris (pH 7.5), 500 mM NaCl, and 2 mM DTT and lysed by sonication. After centrifugation, the clarified cell lysate was incubated with glutathione-sepharose 4B beads. GST-tagged SDC4 was eluted with the buffer consisting of 50 mM reduced glutathione. Protein fractions were collected in buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, and 2 mM DTT and used for the indicated assays.

Drug affinity responsive target stability

According to a previous study (55), SW480 cells were lysed with M-PER (78503, Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors (78420, Thermo Fisher Scientific). After centrifugation (14,000 rpm, 15 min), the supernatant was incubated with AGO or DMSO for 1 h at room temperature. Samples were then digested with diluted pronase (4.2 mg/ml) for another 30 min, and finally terminated by adding 2× SDS loading buffer, followed by heating at 95 °C for 10 min. The samples were analyzed by Western blot.

IP

IP was performed according to the Dynabeads CoImmunoprecipitation Kit protocol (10004D, Invitrogen). Briefly, the protein extracts were incubated with the equilibrated beads at 4 °C overnight with gentle mixing to capture the Flag fusion proteins or specific antibody-captured proteins. The magnetic beads or agarose beads were collected by placing the tube in the appropriate magnetic separator. The beads were washed with PBST buffer to remove all of the non-specifically bound proteins. The bound fusion proteins were eluted from the beads with the corresponding elution buffer for Western blotting analysis. The following are primary antibodies and dilutions used for IP assay: anti-SDC4 (sc12766, 1:100), anti-Flag (F1804, 1:50, Sigma-Aldrich).

Proximity ligation assay

Proximity ligation assay was performed according to the protocol provided by Sigma-Aldrich (DUO092101), and the primary antibodies and dilutions are anti-SDC4 (sc12766, 1:100), anti-Syntenin (22399-1-AP, 1:400, Proteintech).

Flow cytometry

Cells were resuspended in the non-enzymatic cell dissociation buffer (C5789, Sigma). The cell pellet was then resuspended in cold PBS with 5% (w/v) BSA. To measure surface populations of SDC4, resuspended cells (>10,000) were incubated with FITC-conjugated anti-SDC4 or its isotype-matched control antibody (sc-12766, Santa Cruz) for 15 min on ice and processed for flow cytometry analysis following the manufacturer’s instructions. Flow cytometry (CytoFLEX S, Beckman) data were analyzed using CytExpert software (https://www.beckman.hk/flow-cytometry/research-flow-cytometers/cytoflex/software) in accordance with the manufacturer’s specifications.

Quantitative RT-PCR

Total RNA was extracted using TRIzol (T9424, Sigma) following the manufacturer’s instructions. The reverse transcription reaction was performed with the 5 × Evo M-MLV RT Master Mix (AG11706, Accurate Biology). The expression of target genes was measured using the QuantStudio five real-time PCR system (Applied Biosystems) with the Hieff qPCR SYBR Green Master Mix kit (11202ES03, Yeasen). All reactions were conducted in triplicate. Relative quantification was calculated using the 2-ΔCt method, with GAPDH as the internal control. The primer sequences for RT-PCR are listed in Table S1.

sEVs isolation and fluorescent labeling

Conditioned mediums were collected from SW480 cells treated with DMSO or AGO (20 μM) after 30 to 48 h of serum-free culture. The mediums were centrifuged twice (500g for 10 min and 10,000g for 30 min) to remove cell debris and larger cell vesicles, followed by filtration through a 0.22 μm filter. sEVs were extracted from the supernatant through an enhanced sEVs isolation kit (C3622, Beyotime) according to the manufacturer’s instructions. The final pellet was resuspended in PBS for subsequent experiments. sEVs were labeled with PKH67 membrane dye (C3635S, Beyotime) according to the manufacturer’s instructions for tracking. Briefly, 100 μl of PKH67 working solution was used to label approximately 10 μg of sEVs. After incubation at room temperature for 1 to 5 min, an equal volume of stop solution was added. Excessive PKH67 was removed with the Spin Column (P2613, Beyotime).

Nanoparticle tracking analysis

SEVs particle concentration and size distribution were analyzed using the NanoSight NS300 system (Malvern Technologies) with a red laser (638 nm) and sCMOS camera. Samples were diluted in PBS and injected at a constant flow rate. Three 30-s videos were captured at camera gain between 280 and 560, and the shutter speed of 15 or 30 ms. Data was analyzed using nanoparticle tracking analysis software (v2.3) (https://www.malvernpanalytical.com/en/support/product-support/nanosight-range/nanosight-ns300).

Transmission electron microscopy analysis

SW480 cells treated with DMSO or AGO (20 μM) after 24 h were fixed overnight at 4 °C in 2.5% glutaraldehyde (in PBS), post-fixed with 1% osmium tetroxide for 2 h, and dehydrated in a graded ethanol series (50%, 70%, 80%, 90%, and 100%). After acetone treatment, samples were embedded, sectioned using an EMUC7 ultramicrotome (Leica), and stained with uranyl acetate and lead citrate. Sections were observed under a Tecnai G2 Spirit transmission electron microscopy (Thermo FEI) at 80 kV.

In vitro sEVs uptake assay

sEVs derived from DMSO or AGO treated SW480 cells were labeled with PKH67 green fluorescent dye. HeLa cells were seeded on 24-well plates at a density of 5 × 10ˆ4 cells/ml. After overnight incubation, cells were treated with sEVs (2 × 10ˆ8 particles/ml). After 2 h of incubation, cells were fixed with 4% PFA, washed, and labeled with Hoechst for nuclear staining. Images of 16 fields per well and condition were captured using the Operetta CLS High Content Analysis System (PerkinElmer).

Statistical analysis

The statistical details of each experiment, including the statistical methods, the p-value, and sample size (n), are shown in the figure legends. GraphPad Prism seven (https://www.graphpad.com/features) was used to plot all graphs and perform statistical and quantitative analyses. Error bars represent the standard error of the mean.

Data availability

All data pertinent to this work are contained within this manuscript or available upon request. Further inquiries can be directed to the corresponding author.

Supporting information

This article contains supporting information.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.