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.


Introduction
Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant digestive system cancers and represents the seventh leading cause of cancer-related death worldwide (1,2). Accumulating reports have shown that lymph node (LN) metastasis represents the major metastatic route of PDAC and that it predicts extremely poor prognosis, where it decreases the 5-year survival rate of patients who have received pancreatoduodenectomy or distal pancreatectomy from 40% to 10% (3,4). The development of LN metastasis in PDAC requires multiple complex processes, among which lymphangiogenesis, the generation and sprouting of lymphatic vessels from pre-existing lymphatic vasculature, represents the predominant step (5)(6)(7)(8). The current antilymphangiogenesis therapies with monoclonal antibodies, micromolecular peptides, or inhibitors targeting vascular endothelial growth factor (VEGF) signaling, the well-characterized pathway for inducing lymphatic vasculature, have achieved limited efficacy against metastatic PDAC in the past decade, prompting the need for developing therapeutic targets of LN metastatic PDAC (3).
KRAS has been well characterized as a membrane-bound GTPase widely involved in cell growth, migration, and survival (9,10). One-fifth of all human cancers, including 85%-90% of PDAC, harbor KRAS activating mutations (9). The KRAS G12D mutation is the most prevalent mutation among the PDAC-associated KRAS mutations, causing pancreatic duct epithelium transition to focal premalignant ductal lesions and also inducing rapid progression to highly invasive and metastatic PDAC by fostering the hyperactivation of several central cellular growth signaling pathways, including mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and Ras-like GEF (RalGEF) (11,12). KRAS G12D mutation-related activation promotes the aggregation of tumor cells around lymphatic vessels, which has been associated with the presence of LN metastasis in PDAC (13,14). Nonetheless, the precise mechanism of KRAS mutation in PDAC lymphangiogenesis and LN metastasis remains unclear.
Lymph node (LN) metastasis occurs frequently in pancreatic ductal adenocarcinoma (PDAC) and predicts poor prognosis for patients. The KRAS G12D mutation confers an aggressive PDAC phenotype that is susceptible to lymphatic dissemination. However, the regulatory mechanism underlying KRAS G12D mutation-driven LN metastasis in PDAC remains unclear. Herein, we found that PDAC with the KRAS G12D mutation (KRAS G12D 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 KRAS G12D -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 adenylateand 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 KRAS G12D PDAC in a genetically engineered Kras G12D/+ Trp53 R172H/+ 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 KRAS G12D PDAC.

KRAS mutant-driven SUMOylation controls extracellular vesicle transmission to trigger lymphangiogenesis in pancreatic cancer
The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases, which showed that 3 hnRNPs, including hnRNPA1, RALY, and SYNCRIP, were upregulated in PDAC versus nontumorous tissues by more than 2-fold and were correlated with poor prognosis of patients with PDAC ( Figure 1D, Supplemental Figure 1, B-M, and Supplemental Table 1). Further validation in a larger cohort of 186 cases of PDAC patients by both quantitative reverse transcription PCR (qRT-PCR) and Western blotting analysis showed that hnRNPA1 was significantly overexpressed in PDAC and correlated with the KRAS G12D mutation (Figure 1, E-G, Supplemental Figure 1N, and Supplemental Figure 2). Kaplan-Meier curve analysis demonstrated that hnRNPA1 overexpression was associated with shorter overall survival (OS) and disease-free survival (DFS) of patients with KRAS G12D PDAC (Supplemental Figure 3, A and B), indicating that hnRNPA1 is a crucial participant in KRAS G12D PDAC. Accordingly, hnRNPA1 was selected for further analysis.
Clinical relevance analysis revealed that hnRNPA1 was overexpressed in patients with KRAS G12D PDAC with LN metastasis as compared with those without LN metastasis ( Figure 1H and Supplemental Figure 3C). Moreover, we observed a positive correlation between hnRNPA1 expression and microlymphatic vessel density indicated by lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) in both the intratumoral and peritumoral regions of KRAS G12D PDAC tissues (Figure 1, I and J), indicating that hnRN-PA1 is correlated with lymphangiogenesis in KRAS G12D PDAC. Taken together, these findings reveal that hnRNPA1 is associated with lymphangiogenesis and LN metastasis of KRAS G12D PDAC.
HnRNPA1 is enriched in EVs secreted by KRAS G12D PDAC cells. Strikingly, we found that hnRNPA1 existed in the extracellular region of KRAS G12D PDAC tissues (Supplemental Figure 3C). The KRAS G12D PDAC tissues with LN metastasis had higher extracellular hnRNPA1 expression than those without LN metastasis (Supplemental Figure 3C), indicating that hnRNPA1 might facilitate KRAS G12D PDAC LN metastasis in its extracellular form. Given that EVs, the nanoscale carriers for communication between tumor cells and stromal cells, have been considered to mediate molecules crossing the extracellular matrix into lymphatic circulation (21), we isolated the EVs from the culture media of PDAC cells with different KRAS subtypes (KRAS G12D : PANC-1, AsPC-1; KRAS G12V : Capan-2; KRAS G12C : Mia-PaCa-2; KRAS WT : BxPC-3) to investigate whether hnRNPA1 exhibited its function in KRAS G12D PDAC cell-secreted EVs. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) identified cupshaped particles 50 to 130 nm in size ( Figure 1, K and L, and Supplemental Figure 3, D and E). Western blotting analysis revealed a higher expression level of the EV markers ALG-2-interacting protein X (ALIX), CD63, and CD9 in the isolated particles than the cellular lysate, while the cellular marker calnexin was rarely detected in the isolated particles (Supplemental Figure 3F), supporting the idea that the isolated particles were EVs. HnRNPA1 was specifically upregulated in KRAS G12D PDAC cells and the corresponding EVs as compared with PDAC cells with other KRAS subtypes or normal human pancreatic ductal epithelial (HPDE) cells ( Figure 1, M and N, and Supplemental Figure 4, A and B). Since the TME of PDAC is accompanied with highly infiltrated cells, which release abundant EVs into the extracellular space of PDAC tissues, Tumor cell-secreted EVs play an important role in reshaping the tumor microenvironment (TME) by transferring biological molecules to modulate stromal cell metabolism and self-renewal, resulting in tumor metastasis (17,18). The application of fibroblast-like mesenchymal cell-derived EVs for transmitting small interfering RNA (siRNA) specifically targeting the KRAS G12D mutation achieved satisfactory efficacy in inhibiting PDAC progression and now are undergoing phase I/II clinical testing (19). Therefore, elucidating the mechanism of EVs in KRAS mutant-triggered PDAC LN metastasis is of great clinical importance for developing the effective engineering of an EV-dependent therapeutic approach against LN metastatic PDAC.
In the present study, we demonstrated that the KRAS G12D mutation was accompanied by lymphangiogenesis hyperactivation in PDAC, and found that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) was specifically upregulated in KRAS G12D PDAC cell-secreted EVs, which was positively associated with LN metastasis of KRAS G12D PDAC. HnRNPA1 packaged by KRAS G12D PDAC cell-secreted EVs was transmitted to human lymphatic endothelial cells (HLECs) to promote lymphangiogenesis and LN metastasis in vitro and in vivo. Moreover, hnRNPA1 was SUMOylated by KRAS G12D mutation-induced overexpression of SUMOactivating enzyme subunit 1 (SAE1), which triggered EV packaging of hnRNPA1 and its delivery to HLECs and subsequently facilitated KRAS G12D PDAC lymphangiogenesis and LN metastasis. Our results highlight a mechanism by which the KRAS G12D mutation induces lymphangiogenesis and LN metastasis by controlling SUMOylation-related transmission of EV-packaged hnRNPA1 in PDAC, highlighting the possibility that hnRNPA1 may be an attractive therapeutic target in KRAS G12D PDAC.

Results
HnRNPA1 is correlated with LN metastasis in KRAS G12D PDAC. KRAS G12D represents the leading mutation in PDAC and causes tumor cell aggregation around lymphatic vessels, implying that it might be related to tumor metastasis through lymphatic vasculature in PDAC (13). Therefore, the KRAS mutations in our clinical PDAC samples were verified in-house by Sanger sequencing, and analysis of the samples by immunohistochemistry (IHC) showed an increase in microlymphatic vessels in the KRAS G12D PDAC tissues as compared with cancer tissues with other KRAS subtypes ( Figure 1, A-C). As lymphatic vessel expansion is conducive to tumor cell metastasis to the LNs, we analyzed the correlation between KRAS G12D and LN metastasis of PDAC. A higher rate of LN metastasis was observed in PDAC with KRAS G12D mutation than in PDAC with other KRAS subtypes, suggesting that the KRAS G12D mutation was associated with LN metastasis of PDAC (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI157644DS1). Given that we and others have revealed that the majority of cancerassociated RNAs trigger tumor lymphangiogenesis by interacting with RNA-binding proteins (RBPs), among which hnRNPs were previously demonstrated to be the specific type of RBPs that correlated with various tumor LN metastasis (5,20), we investigated the hnRNPs that contributed to KRAS G12D -associated lymphangiogenesis and LN metastasis in PDAC. First, the screening of hnRNPs was performed in PDAC and nontumorous tissues from and Supplemental Figure 4, O and P). These results demonstrate that EV-packaged hnRNPA1 secreted by KRAS G12D PDAC cells facilitates the tube formation and migration of HLECs to induce lymphangiogenesis in vitro.
EV-packaged hnRNPA1 induces LN metastasis of KRAS G12D PDAC in vivo. To explore whether hnRNPA1 was involved in KRAS G12Dinduced LN metastasis of PDAC in vivo, we established the popliteal lymphatic metastasis model through implanting hnRNPA1-overexpressing or -knockdown PANC-1 cells (KRAS G12D ) or BxPC-3 cells (KRAS WT ) and corresponding control cells separately. HnRNPA1 overexpression significantly promoted PANC-1 cell metastasis to the popliteal LNs and hnRNPA1 knockdown suppressed the LN metastasis of PANC-1 cells, as indicated by an in vivo imaging system (IVIS), while the alteration of hnRNRA1 expression in BxPC-3 produced only rare effects on LN metastasis (Supplemental Figure 5A). Larger LNs were detected in the hnRNPA1-overexpressing PANC-1 group as compared with the control PANC-1 group, whereas decreased LN volumes were detected in the hnRN-PA1-knockdown group (Supplemental Figure 5B). Moreover, the microlymphatic vessel density in primary tumors was dramatically increased by hnRNPA1 overexpression and reduced by hnRNPA1 knockdown, while either hnRNPA1 overexpression or knockdown in BxPC-3 only slightly affected the quantification of microlymphatic vessels (Supplemental Figure 5C), indicating that hnRNPA1 is involved in KRAS G12D -induced LN metastasis of PDAC.
As we indicated that hnRNPA1 fostered the lymphangioge nesis of KRAS G12D PDAC through the EV-packaged form, we further evaluated the effect of EV-packaged hnRNPA1 on LN metastasis of KRAS G12D PDAC in an EV-induced popliteal lymphatic metastasis model ( Figure 3A). Subsequently, the mice were intratumorally treated with PBS, EVs secreted by PDAC cell lines with different KRAS subtypes (KRAS WT : BxPC-3-EV Vector ; KRAS G12V : Capan-2-EV Vector ; KRAS G12D : PANC-1-EV Vector ), or EVs secreted by hnRNPA1-overexpressing PANC-1 cells (PANC-1-EV hnRNPA1 ) (Supplemental Figure 5, D-F). IVIS showed that PANC-1-EV hnRNPA1 significantly promoted PANC-1 cell metastasis to the popliteal LNs when compared with the PANC-1-EV Vector , while treatment with PBS or EVs secreted by PDAC cell lines with other KRAS subtypes (BxPC3-EV Vector or Capan-2-EV Vector ) had only rare effects on the popliteal LN metastasis of mice ( Figure 3, B and C, and Supplemental Figure 5, G-I). Moreover, the PANC-1-EV hnRNPA1 group had significantly increased the popliteal LN volumes, while PANC-1-EV Vector slightly enlarged the popliteal LNs when compared with the PBS, BxPC3-EV Vector , and Capan-2-EV Vector groups ( Figure 3, D and E, and Supplemental Figure 5J). Increased LN metastatic rates were observed in mice treated with PANC-1-EV hnRNPA1 as compared with those that received PANC-1-EV Vector treatment (Supplemental Figure 5K). Importantly, confocal microscopy revealed significant internalization of PKH67labeled EVs by lymphatic vessels in the PANC-1-EV hnRNPA1 group, which increased the number of microlymphatic vessels as indicated by representative markers of lymphangiogenesis, including LYVE-1, podoplanin, VEGFR3, CD31, and NRP2 in the intratumoral and peritumoral regions of the primary tumors. Since infiltrated cells in the TME have been previously reported to contribute to the lymphangiogenesis and promote LN metastasis (22, 23), we evaluated whether the abundant cells in the TME of PDAC, we also evaluated the expression of hnRNPA1 in EVs secreted by the predominant cells in the TME of PDAC, including fibroblasts, macrophages, T cells, and B cells, as well as the tumor cells. The results showed that hnRNPA1 expression was significantly higher in EVs from KRAS G12D PDAC cells compared with EVs secreted by the other cells in the TME (Supplemental Figure 4, C and D), indicating that hnRNPA1 is predominantly enriched in EVs secreted by KRAS G12D PDAC cells.

EV-packaged hnRNPA1 secreted by KRAS G12D PDAC cells enhances tube formation and migration of HLECs in vitro.
Considering that lymphangiogenesis represents the determinant process mediating lymphatic dissemination of PDAC cells to the draining LNs and fosters LN metastasis, we explored the role of EV-packaged hnRNPA1 in the tube formation and migration of HLECs in vitro. EVs secreted by PANC-1 and ASPC-1 (KRAS G12D ) cells with higher hnRNPA1 expression levels markedly promoted HLEC tube formation and migration as compared with the control ( Figure  2, A-C). HnRNPA1 knockdown in the KRAS G12D PDAC cells was followed by decreased hnRNPA1 expression levels in the corresponding EVs and hnRNPA1 overexpression induced hnRNPA1 enrichment in the KRAS G12D PDAC cell-secreted EVs, while the expression levels of hnRNPA1 in EVs changed slightly after altering the cellular hnRNPA1 expression in PDAC cells with other KRAS subtypes (   and quantification of tube formation and migration (B and C) for HLECs treated with PBS or PDAC cell-secreted EVs. Scale bar: 100 μm. One-way ANOVA followed by Dunnett's test was used. (D and E) Western blotting analysis of hnRNPA1 protein levels in PANC-1 cell-secreted EVs after hnRNPA1 silencing or overexpression. (F and G) Representative images and quantification of tube formation and migration by HLECs treated with PBS or indicated EVs. Scale bars: 100 μm. One-way ANOVA followed by Dunnett's test was used. Data are presented as mean ± SD of 3 independent experiments. *P < 0.05, **P < 0.01. KRAS G12D PDAC independent of the infiltrated cells, including CAFs and TAMs in the TME. Together, our results demonstrate that EV-packaged hnRNPA1 induces KRAS G12D PDAC lymphangiogenesis and LN metastasis.
To simulate the anatomy and physiology of LN metastasis in vivo, we established an orthotopic xenograft model to investigate the role of EV-packaged hnRNPA1 in LN metastasis of KRAS G12D PDAC ( Figure 3I). Positron emission tomography-computed tomography (PET-CT) scanning showed that the PANC-1-EV hnRNPA1 group had higher accumulation of 18 F-fluorodeoxyglucose ( 18 FDG) than the PANC-1-EV Vector group (Figure 3, J and K, and Supplemental Figure  6A), suggesting that EV-packaged hnRNPA1 promoted the orthotopic tumorigenicity of KRAS G12D PDAC cells. Given that the peripancreatic LNs in the abdomen, including the pyloric, hilar, and superior mesenteric LNs, represent the most common drainage LNs of PDAC in mice (24), we enucleated them to evaluate the effects of EV-packaged hnRNPA1 on LN metastasis of KRAS G12D PDAC. The overexpression of EV-packaged hnRNPA1 significantly facilitated PANC-1 cell metastasis to the peripancreatic LNs (Supplemental Figure 6, B-E, and Supplemental Table 2). Furthermore, PANC-1-EV hnRNPA1 treatment promoted lymphangiogenesis in the primary tumor and the subcapsular sinus of the peripancreatic LNs ( Figure 3, L-N, and Supplemental Figure 6, F and G). Additionally, only rare differences in metastasis to the liver or omentum was found between the PANC-1-EV Vector and PANC-1-EV hnRNPA1 groups (Supplemental Figure 6, H and I), suggesting the specific role of EV-packaged hnRNPA1 in LN metastasis rather than distant metastasis. Collectively, these findings demonstrate that EV-packaged hnRNPA1 promotes KRAS G12D PDAC lymphangiogenesis and LN metastasis in vivo.
KRAS signaling-induced SAE1 overexpression catalyzes hnRN-PA1 SUMOylation. As we indicated that EV-packaged hnRNPA1 overexpression induced lymphangiogenesis and LN metastasis of KRAS G12D PDAC, we explored the molecular mechanism triggering hnRNPA1 enrichment in KRAS G12D PDAC cell-secreted EVs. Interestingly, we found that EV-packaged hnRNPA1 had a higher molecular weight (>40 kDa) when compared with the hnRNPA1 in the cells (<40 kDa) ( Figure 4A), suggesting that hnRNPA1 in KRAS G12D PDAC cell-secreted EVs underwent posttranslational modification (PTM). Then, we used inhibitors targeting various PTMs to detect the vital PTM involved in the high hnRNPA1 enrichment in KRAS G12D PDAC cell-secreted EVs. Only 2-D08, a specific inhibitor of SUMOylation, significantly decreased hnRN-PA1 expression levels in the PDAC cell-secreted EVs, while hnRN-PA1 expression in the PDAC cells was only slightly increased (Figure 4, B and C). Mass spectrometry (MS) analysis of the hnRNPA1 coimmunoprecipitation (co-IP) products showed that 2-D08 significantly suppressed the attachment of SUMO2, a SUMOylation modifier, to hnRNPA1 (Supplemental Figure 7, A and B), which was validated by Western blotting analysis ( Figure 4D). Moreover, SUMO2 knockdown greatly downregulated hnRNPA1 expression levels in the PDAC cell-secreted EVs ( Figure 4E). These results suggest that SUMO2 modification of hnRNPA1 is essential for hnRNPA1 loading into EVs.
Next, we investigated the mechanism triggering hnRNPA1 SUMOylation in KRAS G12D PDAC cells. Accumulating evidence has demonstrated that the KRAS G12D mutation predominantly causes the rapidly accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular regulated protein kinase (RAF/MEK/ERK) signaling pathway to promote PDAC progression (25,26). Accordingly, we used a small-molecule inhibitor targeting the KRAS/ RAF signaling pathway, MCP110, to evaluate whether KRAS G12Dinduced RAF signaling activation stimulates hnRNPA1 SUMOylation in KRAS G12D PDAC cells ( Figure 4F). MCP110 significantly reduced RAF and MEK1/2 phosphorylation without affecting the total levels of RAF and MEK1/2 ( Figure 4G), suggesting the successful inhibition of the KRAS/RAF signaling pathway. Among the multiple SUMOylation-related enzymes, the expression of SAE1, the crucial E1 SUMO-activating enzyme for SUMOylation modification (27), was significantly decreased after MCP110 treatment in the KRAS G12D PDAC cells (Figure 4, H-J). Moreover, overexpressing SAE1 significantly promoted SUMO2 modification of hnRNPA1 and facilitated hnRNPA1 packaging into the EVs (Figure 4, K and L). The in vitro experiments showed that SAE1 overexpression enhanced the abilities of PDAC-secreted EVs to induce HLEC tube formation and migration, which was reversed by downregulating hnRNPA1 expression in the PDAC-secreted EVs (Figure 4, M-O). Collectively, these findings demonstrate that the KARS G12D mutation upregulated SAE1 expression to induce the SUMOylation and EV sorting of hnRNPA1.
SUMOylation of hnRNPA1 enables its packaging into EVs by interacting with TSG101. Since the interactions between proteins contribute to their subcellular location and extracellular exportation (29), we determined the binding partner of SUMOylated hnRNPA1. Co-IP assays followed by silver staining detected an obvious band of 44-55 kDa enriched by hnRNPA1 co-IP in PDAC cells treated with negative control siRNA compared with SAE1-depleted PDAC cells, which MS and Western blotting analyses identified as tumor susceptibility 101 (TSG101) (Figure 6, A and B, and Supplemental Figure 8, A and B). SAE1 overexpression promoted hnRNPA1's interaction with TSG101, which was critically inhibited by the hnRNPA1 K113R mutation ( Figure 6C), confirming that SAE1-induced SUMOylated hnRNPA1 bound directly with TSG101. Moreover, hnRNPA1 and TSG101 were colocalized in the nuclei of PDAC cells ( Figure 6D). As TSG101 is a crucial component of the endosomal sorting complex responsible for transport (ESCRT) and triggers EV synthesis by loading proteins into EV precursors (30, 31), we evaluated whether it EV-packaged hnRNPA1 is delivered to HLECs to induce lymphangiogenesis. Since our results indicated that SUMOylated hnRN-PA1 was packaged into EVs via interaction with TSG101 and subsequently promotes KRAS G12D PDAC lymphangiogenesis, we investigated how EV-packaged hnRNPA1 regulated HLECs. PDAC cell-secreted EVs were labeled with PKH67 and incubated with HLECs. Confocal microscopy revealed that the green fluorescence signal from the PKH67-labeled EVs was present in the HLEC cytoplasm, while no such signal was detected in the control group ( Figure 7A). Moreover, HLECs treated with PANC-1-EVsi-hnRNPA1#1 (PANC-1 cell EVs with hnRNPA1 silencing) exhibited lower hnRNPA1 expression levels than the control group, while hnRNPA1 overexpression was detected in HLECs treated with mediated hnRNPA1 packaging into EVs. TSG101 knockdown significantly decreased hnRNPA1 enrichment in PDAC cell-secreted EVs without affecting cellular hnRNPA1 expression, while hnRNPA1 was significantly upregulated in EVs secreted by TSG101-overepressing cells (Figure 6, E and F, and Supplemental Figure 8, C and D), suggesting that TSG101 promoted hnRNPA1 packaging into EVs. Furthermore, we assessed whether TSG101 was essential for EV transmission of hnRNPA1 for inducing lymphangiogenesis in PDAC. The results showed that TSG101 knockdown greatly inhibited EV-packaged-hnRNPA1-induced HLEC tube formation and migration ( Figure 6, G and H). Altogether, these findings demonstrate that SUMOylation on hnRNPA1 K113 triggers its packaging into EVs with the assistance of TSG101 in KRAS G12D PDAC. These results are consistent with those obtained in wildtype hnRNPA1 (hnRNPA1 WT ) HLECs in vitro, suggesting that PDAC-secreted EVs regulated HLEC function by transmitting EV-packaged hnRNPA1 rather than by activating hnRNPA1 transcription. Taken together, our findings demonstrate that KRAS G12D PDAC cell-secreted EVs induce lymphangiogenesis by delivering EV-packaged hnRNPA1 to HLECs.
SUMOylation of EV-packaged hnRNPA1 enhances prospero homeobox 1 mRNA stability in HLECs. It has been proposed that VEGF-C PANC-1-EV hnRNPA1 (Figure 7, B and C), indicating that EV-packaged hnRNPA1 had been delivered to the HLECs.
To exclude the possibility that KRAS G12D PDAC cell-secreted EVs promoted HLEC tube formation and migration by inducing endogenous hnRNPA1 transcription in HLECs, we utilized the clustered regularly interspaced short palindromic repeats/CRIS-PR-associated protein 9 (CRISPR/Cas9) approach to construct an endogenous hnRNPA1-knockout (hnRNPA1 KO ) HLEC line (Figure 7, D and E). EV-packaged-hnRNPA1 knockdown suppressed the tube formation and migration of hnRNPA1 KO HLECs induced by PDAC cell-secreted EVs, while EV-packaged-hnRNPA1 Prospero homeobox 1 (PROX1) is considered a key player in lymphatic endothelium maintenance and facilitates lymphatic vessel development during lymphangiogenesis (5,6). Therefore, we investigated PROX1 expression in EV-packaged-hnRNPA1-treated HLECs. The results showed that PROX1 expression correlated positively with hnRNPA1 expression levels in the KRAS G12D PDAC cell-secreted EVs, while EVs secreted by hnRNPA1-overexpressing PDAC cells with other KRAS subtypes or the stromal cells only rarely affected PROX1 expression in HLECs (Figure 8, A-D, and Supplemental Figure 10A), suggesting that PROX1 was the downstream target of EV-packaged hnRNPA1 secreted by KRAS G12D PDAC cells. Dual-luciferase assays for determining the molecular mechanism of EV-packaged hnRNPA1 in regulating PROX1 expression showed that EV-packaged hnRNPA1 had little effect on the PROX1 promoter region, while a significant increase in luciferase activity was observed when activating the PROX1 3′-untranslated region represents the core regulator for inducing tumor lymphangiogenesis (32). Accordingly, we analyzed whether hnRNPA1 participates in regulating VEGF-C to promote the lymphangiogenesis of PDAC. The results showed that either overexpression or knockdown of hnRN-PA1 affected the VEGF-C expression and secretion of PDAC cells (Supplemental Figure 9, A-D). Since VEGFR3 in HLECs has been well characterized as the receptor for VEGF-C to induce the sprouting of lymphatic vessels (33), we further constructed CRISPR/Cas9mediated VEGFR3-knockout HLECs to analyze whether EV-packaged hnRNPA1 triggered lymphangiogenesis independent of VEGF-C signaling (Supplemental Figure 9E). The tube formation and migration of HLECs were significantly inhibited after VEGFR3 knockout, while EV-packaged-hnRNPA1 overexpression still promoted the tube formation and migration of VEGFR3-knockout HLECs (Supplemental Figure 9, F-H), suggesting that hnRNPA1 promotes lymphangiogenesis and LN metastasis independent of VEGF-C. The hnRNPA1 K113R mutation significantly impaired the hnRNPA1induced tube formation and migration of HLECs with or without SAE1 overexpression (Supplemental Figure 11, D-F). Moreover, a popliteal LN metastasis mouse model was constructed to show that EV-packaged-hnRNPA1 overexpression enhanced LN metastasis induced by PDAC-cell-secreted EVs. Downregulating SAE1 to suppress EV-packaged-hnRNPA1 transmission reversed these effects after αVEGF-C treatment in both groups (Figure 9, D and E). Compared with the PANC-1-EV hnRNPA1 plus αVEGF-C group, the PANC-1-EV hnRNPA1+si-SAE1#1 plus αVEGF-C group had reduced incidence of LN metastasis ( Figure 9F). Blocking SUMOylation on hnRNPA1 through SAE1 knockdown also inhibited the EV-packaged-hnRNPA1-induced increase in LYVE-1-positive microlymphatic vessels and PROX1 expression in primary tumors in a VEGF-C-independent manner (Figure 9, G-I). Furthermore, mice in the PANC-1-EV hnRNPA1+si-SAE1#1 plus αVEGF-C group had prolonged survival time compared with those in the PANC-1-EV hnRNPA1 plus αVEGF-C group ( Figure 9J).
Kras G12D/+ Trp53 R172H/+ Pdx-1-Cre (KPC) mice are well characterized as a genetically engineered PDAC model system with autonomously growing tumors to mimic KRAS G12D mutationinduced PDAC progression (35). Therefore, we evaluated the effect of SUMOylation of EV-packaged hnRNPA1 on the regulation of PROX1 expression to induce LN metastasis of KRAS G12D PDAC in the KPC mouse model. The results showed that EVs overexpressing hnRNPA1 significantly promoted LN metastasis in KPC mice and the effect was reversed by inhibiting SAE1induced SUMOylation, while only rare effects on liver or omentum metastasis were observed among these 3 groups (Figure 9, K and L, and Supplemental Figure 11, G-I). IHC analysis revealed that EV-packaged hnRNPA1 increased the LYVE-1-positive microlymphatic vessels and PROX1 expression in primary tumors, which was abolished by SAE1 knockdown (Figure 9, M and N). Taken together, these results indicate that EV-packaged hnRNPA1 promotes lymphangiogenesis and LN metastasis of KRAS G12D PDAC by upregulating PROX1 expression.
The clinical relevance of EV-packaged hnRNPA1 in patients with LN metastatic PDAC. As EV-packaged molecules have been identified as potential biomarkers and therapeutic targets in various cancers (36), we evaluated the clinical relevance of EV-packaged hnRNPA1 in KRAS G12D PDAC at 2 independent clinical centers (96 patients from Sun Yat-Sen Memorial Hospital of Sun Yat-sen University, and 76 patients from Guangdong Provincial People's Hospital). EVs were extracted from the serum samples of patients with KRAS G12D PDAC and healthy controls, which were identified by TEM and NTA analysis (Supplemental Figure 12, A and B). EV-packaged hnRNPA1 was overexpressed in serum EVs from the patients with KRAS G12D PDAC as compared with the healthy controls (Supplemental Figure 12, C-E). Kaplan-Meier survival analysis revealed that EV-packaged-hnRNPA1 expression levels correlated positively with poor prognosis in the patients (Supplemental Figure  12, F-K). Univariate and multivariate analyses identified EV-packaged hnRNPA1 as an independent prognostic factor of OS and DFS of PDAC patients (Supplemental Tables 3 and 4). Moreover, the patients with LN metastasis or advanced tumor stage had higher serum EV-packaged hnRNPA1, SAE1, and PROX1 expression levels (Supplemental Figure 12, L-P, and Supplemental Table 5).
(3′-UTR) (Supplemental Figure 10, B-E). Actinomycin assays also revealed a positive correlation between EV-packaged hnRNPA1 expression levels and the half-life of PROX1 mRNA (Figure 8, E and F, and Supplemental Figure 10F), suggesting that EV-packaged hnRNPA1 upregulated PROX1 expression by stabilizing PROX1 mRNA rather than by affecting PROX1 transcription activity. As KRAS G12D PDAC cell-secreted EV-packaged hnRNPA1 was predominantly SUMOylated, we used SUMO-specific peptidase 3 (SENP3) to inhibit hnRNPA1 SUMOylation in KRAS G12D PDAC cells, which significantly attenuated the ability of EV-packaged hnRNPA1 to stabilize PROX1 mRNA (Figure 8, G and H, and Supplemental Figure  10G). Moreover, the hnRNPA1 K113R mutation significantly impaired EV-packaged-hnRNPA1-induced stabilization of PROX1 mRNA (Figure 8, G and H, and Supplemental Figure 10G), validating that the SUMOylation of EV-packaged hnRNPA1 promoted its effect on PROX1 mRNA stability. Given that the adenylate-and uridylaterich (AU-rich) elements (AREs) in the mRNA 3′-UTR are common determinants of RNA stability in mammalian cells (34), we analyzed whether EV-packaged hnRNPA1 regulated PROX1 mRNA stability via interaction with PROX1 AREs. RNA IP (RIP) showed that EV-packaged hnRNPA1 bound directly to PROX1 mRNA, which was abolished by inhibiting hnRNPA1 SUMOylation (Supplemental Figure 10, H and I). AREsite2 analysis led to the identification of an AU-rich region that contains 3 AUUUA core pentamers in the PROX1 3′-UTR ( Figure 8I). Dual-luciferase reporter assays revealed that EV-packaged hnRNPA1 increased PROX1 promoter luciferase activity via SUMOylation, while inducing mutation in the PROX1 AREs abolished the effects of EV-packaged hnRNPA1 on the PROX1 promoter luciferase activity ( Figure 8J and Supplemental Figure 10J), suggesting that EV-packaged hnRNPA1 interacted directly with the PROX1 AREs. Moreover, the actinomycin assays demonstrated that ARE mutations inhibited the effect of EV-packaged hnRNPA1 on PROX1 mRNA stability (Figure 8, K and L, and Supplemental Figure 10K).
EV-packaged hnRNPA1 promotes PROX1-induced lymphangiogenesis and LN metastasis. As we determined that EV-packaged hnRNPA1 targeted HLECs to enhance PROX1 mRNA stability, we investigated whether PROX1 was required for EV-packaged-hnRNPA1-induced lymphangiogenesis and LN metastasis. The in vitro assays revealed that reducing EV-packaged-hnRNPA1 expression levels abolished HLEC tube formation and migration induced by KRAS G12D PDAC cell-secreted EVs, while PROX1 overexpression reversed this effect even after VEGF-C had been blocked with VEGF-C-neutralizing antibody (αVEGF-C) (Figure 9, A-C). Conversely, PROX1 knockdown reversed EV-packaged-hnRNPA1induced lymphangiogenesis in a VEGF-C-independent manner, indicating that EV-packaged hnRNPA1 facilitated lymphangiogenesis by upregulating PROX1 in HLECs independent of VEGF-C (Supplemental Figure 11, A-C).
Given that SUMOylation-driven EV transmission of hnRNPA1 was conducive to PDAC-secreted-EV-mediated PROX1 overexpression for triggering lymphangiogenesis, we explored whether it contributed to KRAS G12D PDAC LN metastasis. In vitro experiments revealed that ectopic hnRNPA1 expression in HLECs only slightly promoted the tube formation and migration of HLECs, while upregulating SAE1 to induce the SUMOylation of hnRN-PA1 significantly triggered HLEC tube formation and migration. characteristic (ROC) analysis revealed that EV-packaged hnRN-PA1 exhibited superior diagnostic performance for KRAS G12D PDAC when compared with carcinoembryonic antigen (CEA) and carbohydrate antigen 72-4 (CA72-4), as indicated by the area under Patients with higher EV-packaged-hnRNPA1 expression levels had upregulated SAE1 and PROX1 expression that was accompanied by increased microlymphatic vessel numbers ( Figure 10, A-C, and Supplemental Figure 12, Q and R). Importantly, receiver operating Discussion KRAS mutations are identified in more than 90% of patients with PDAC and tend to be associated with advanced stage and reduced OS of PDAC (9). There is increased physical interaction between tumor cells and endothelial cells in KRAS G12D PDAC, which might affect lymphangiogenesis and LN metastasis (13). However, the the curve ( Figure 10D and Supplemental Figure 12S). EV-packaged hnRNPA1 was more effective for distinguishing LN-positive from LN-negative KRAS G12D PDAC than CA19-9, CEA, and CA72-4 ( Figure 10, E and F). Our findings suggest that EV-packaged hnRN-PA1 is a potential biomarker and therapeutic target in LN metastasis of KRAS G12D PDAC. One-way ANOVA followed by Dunnett's test was used. (L) Quantification of the metastatic number of peripancreatic LNs. One-way ANOVA followed by Dunnett's test was used. (M and N) Quantification of IHC analysis for LYVE-1-positive lymphatic vessels and PROX1 expression in pancreatic tumors. Oneway ANOVA followed by Dunnett's test was used. Data are presented as mean ± SD of 3 independent experiments. *P < 0.05, **P < 0.01.
Lymphangiogenesis is well characterized as an essential step in LN metastasis in various cancers (37). Clinical evidence has shown that a high density of lymphatic vessels in PDAC is associated with increased LN metastasis and decreased OS (38,39). Currently, the universally acknowledged mechanism for lymphangiogenesis mainly focuses on the VEGF-C-mediated lymphatic pathways (3,40). Nevertheless, VEGF-C-targeted therapy fails to achieve satisfactory efficacy in 30% of PDAC with LN metastasis, encouraging further elucidation of the mechanism of lymphangiogenesis independent of VEGF-C in PDAC (3). Herein, we showed that lymphangiogenesis and LN metastasis occurred more frequently in KRAS G12D PDAC. KRAS G12D PDAC cells directly targeted PROX1 mRNA in HLECs by transmitting SUMOylated hnRNPA1 in a VEGF-C-independent manner, after which SUMOylated hnRNPA1 directly bound to the PROX1 ARE region to enhance PROX1 mRNA stability, thereby mechanism by which the KRAS G12D mutation regulates LN metastasis of PDAC remains unclear. In the present study, we uncovered that hnRNPA1 was upregulated in KRAS G12D PDAC cell-secreted EVs and promoted EV-mediated lymphangiogenesis and LN metastasis in both in vitro experiments and in xenografted, genetically engineered KPC mouse models. Moreover, hnRNPA1 was bound to SUMO2 as a result of KRAS G12D -induced SAE1 overexpression, which enhanced its physical interaction with TSG101 and triggered EV transmission of hnRNPA1. Subsequently, EV-packaged SUMOylated hnRNPA1 upregulated PROX1 expression in HLECs by stabilizing PROX1 mRNA to facilitate the lymphangiogenesis of KRAS G12D PDAC. Our study clarifies a mechanism underlying KRAS mutant-related lymphangiogenesis and LN metastasis in PDAC through the induction of SUMOylation-related EV transmission, providing a perspective on clinical interventions for LN metastasis of KRAS G12D PDAC. triggered the delivery of the aforementioned EVs into the TME to remodel the lymphatic vasculature. Blocking SAE1 abolished EV transmission of hnRNPA1 and inhibited PDAC LN metastasis in KPC mouse models. The identification of the machinery underlying KRAS mutant-driven SAE1-induced SUMOylation and its role in regulating EV-packaged-hnRNPA1-mediated lymphangiogenesis suggests that SAE1-mediated hnRNPA1 SUMOylation might represent a promising target for therapeutic strategies for suppressing KRAS-related LN metastasis of PDAC.
Another important finding was the improvement of LN metastasis diagnosis with the application of EV-packaged hnRNPA1. Currently, the assessment of LN status of PDAC mainly relies on imaging-based approaches, which are inaccurate, especially for early lesions (50). Therefore, monitoring LN metastasis in PDAC remains greatly challenging. Recently, there has been increased research attention on EV-packaged molecules because of their clinical significance as a convenient and noninvasive indicator in cancer diagnosis and risk stratification (15,(51)(52)(53). Herein, we found that EV-packaged hnRNPA1 was upregulated in the serum EVs from patients with PDAC and correlated positively with LN metastasis. Moreover, EV-packaged-hnRNPA1 expression levels exhibited greater accuracy than CEA or CA72-4 for differentiating patients with KRAS G12D PDAC from healthy controls, and had similar accuracy to that of CA19-9. Moreover, the detection of EV-packaged hnRNPA1 effectively distinguished patients with KRAS G12D PDAC with LN metastasis from those without LN metastasis, highlighting that EV-packaged-hnRNPA1 expression levels might be a feasible biomarker for overcoming the challenge of diagnosing LN metastasis in PDAC.
In summary, our findings provide essential information on the mechanism underlying KRAS-related regulation of lymphangiogenesis through the transmission of EV-packaged hnRNPA1 in a SUMOylation-dependent manner. Moreover, we found a positive correlation between EV-packaged hnRNPA1 and LN metastasis in patients with KRAS G12D PDAC and demonstrate its potential application in the clinical assessment of LN metastasis. Finally, our study highlights the role of KRAS-mutant-driven SUMOylation in triggering the delivery of EV-packaged hnRNPA1 to facilitate lymphangiogenesis. These results suggest hnRNPA1 as a potential therapeutic target for LN metastasis in KRAS G12D PDAC.

Methods
Patient samples. A total of 186 patients with PDAC who had undergone surgery at Sun Yat-Sen Memorial Hospital of Sun Yat-sen University and another 76 patients with KRAS G12D mutation who had undergone surgery at Guangdong Provincial People's Hospital were included. All PDAC tissues, confirmed by 2 pathologists independently, and paired normal adjacent tissues were acquired and quickly frozen in liquid nitrogen for protein extraction, or formalin-fixed and paraffin-embedded for IHC analysis. Blood samples were obtained from the patients with KRAS G12D PDAC and 172 paired healthy participants at the 2 independent centers.
Cell lines and cell culture. Human PDAC cell lines (KRAS G12D : PANC-1, AsPC-1; KRAS G12V : Capan-2; KRAS G12C : Mia-PaCa2; KRAS WT : BxPC-3) were obtained from American Type Culture Collection (ATCC). HPDE cells were obtained from Binsui Biotechnology. The HLECs were obtained from ScienCell Research Laboratories. The PANC-1 (ATCC, CRL-1469MET; RRID: CVCL_A4BT) and Capan-2 cells (ATCC, HTB-80; RRID: CVCL_0026) were maintained in DMEM promoting PDAC lymphangiogenesis. These findings demonstrate the VEGF-C-independent mechanism underlying LN metastasis of KRAS G12D PDAC by which SUMOylated hnRNPA1 regulates PROX1 mRNA stability via EV transmission to induce lymphangiogenesis. In addition, accumulating evidence revealed that engineered EVs represent a prospective approach with high histocompatibility and targeted capacity for cancer therapy (41). Since mRNA stability is an important posttranscriptional regulatory process that allows rapid adjustment of the PROX1 mRNA copy number and is crucial for driving the response of LECs (42), our results provide evidence for the potential application of PROX1-targeted engineered EVs in the treatment of LN metastatic PDAC.
EVs acquire various biological functions by packaging specific molecules during their biogenesis (43). It has been proposed that molecule packaging requires recognition by the ESCRT (44). The ESCRT consists of ESCRT-0, -I, -II, -III, and Vps4 complexes, directing protein incorporation into the endocytic system and the subsequent membrane abscission away from the cytosol to produce EVs (44,45). ESCRT component activation and dysregulation alter EV contents and behaviors (46). However, the core regulator of ESCRT and its role in EV-induced PDAC LN metastasis remain unexplored. Herein, we found that TSG101 was specifically recruited by the lymphangiogenesis-driven protein hnRNPA1 and subsequently guided its transmission via EVs. Blocking SUMOylation eliminated TSG101-mediated encapsulation of EV-packaged hnRNPA1 and significantly suppressed the lymphangiogenesis and LN metastasis of PDAC both in vitro and in vivo. Additionally, it has been reported that hnRNPA1 participates in the sorting of RNAs into EVs to affect the various biological features of cancer (47,48). Nevertheless, we found that treating simply with ectopic hnRNPA1 after the induction of its SUMOylation was able to facilitate lymphangiogenesis, implying that TSG101-induced EV transmission of SUMOylated hnRNPA1 represents a distinct mechanism independent of the role of hnRNPA1 in mediating the biomolecule transmission by EVs. These findings support the crucial role of the TSG101-dependent EV sorting pathway in PDAC lymphangiogenesis, suggesting a potential strategy for blocking EV transmission to suppress LN metastasis of PDAC.
SUMOylation represents a common biological event in protein regulation that affects protein stability, subcellular localization, or interaction ability (28,49). Previously, we reported that SUMOylation induced by UBC9, the E2 ligase of SUMOylation, contributed to tumor lymphangiogenesis (6). Here, we identified that, in KRAS G12D PDAC, activation of KRAS signaling predominantly induced the SUMOylation pathway by upregulating SAE1 rather than UBC9, suggesting that SAE1 exhibits a more prominent function in KRAS mutation-induced SUMOylation to facilitate PDAC progression. As the most abundant E1 SUMO-activating enzyme in cancer, SAE1 initiates SUMOylation modification by catalyzing the C-terminal adenylation of SUMOs (28,49). Binding with SUMOs mediates protein or RNA extracellular delivery, which induces cells in the TME to form a supportive environment for tumor metastasis (27). However, the role of SAE1 in triggering SUMOylation-mediated regulation of the TME to facilitate PDAC progression is largely unexplored. In the present study, we reported that the SAE1 overexpression induced by the KRAS/RAF signaling pathway sustained the SUMOylation of hnRNPA1 and triggered its packaging into EVs. Subsequently, it