Protein-protein interaction network and cell model construction
To identify the proteins that interact with LOXL2, we performed co-IP using LOXL2 as bait followed by LC-MS analysis. Mass spectrometry results showed that multiple LCN2 protein peptides were identified from the differentially-expressed 25kD band in LOXL2-overexpressing cells. The PEP values of the experimental group peptides were far less than 0.1, and the cumulative coverage was as high as 42.3%, indicating the results were highly credible (Supplementary Table 2). The secondary ion mass spectrum of the VPLQQNFQDNQFQGK proteolysis product of LCN2 protein is shown in Supplementary Fig. S1A. The peaks represented the secondary ion peaks of the peptide measured by the mass spectrum. These results indicated that LOXL2 and LCN2 interacted to form a complex.
Next, to gain a complete view of the interactome between LCN2 and LOXL2, all literature reporting interacting proteins was obtained from HPRD (http://www.hprd.org/) and BioGRID (http://thebiogrid.org/), and were processed and merged into a "parent network" as previously described . The parental network was entered into the Cytoscape program to construct a secondary protein interaction sub-network using LCN2 and LOXL2 as the seed proteins. LCN2 was linked with LOXL2 by at least 5 pair partner proteins, further supporting that LCN2 interacts directly or physically with LOXL2 (Supplementary Fig. S1B). To understand their correlation in ESCC clinical samples, the Pearson correlations for LCN2/MMP9, LCN2/LOXL2 and MMP9/LOXL2 in the public GSE53625 dataset were examined. A significant correlation was found for LCN2/MMP9 and MMP9/LOXL2 (Supplementary Fig. S1C).
The endogenous expression level of LCN2 and LOXL2 was examined in eight esophageal cancer cell lines, with human 293T as a control. KYSE410 and KYSE150 cells showed relatively low endogenous LCN2 and LOXL2 levels, and were selected as the cell model for subsequent experiments (Supplementary Fig. S1D). KYSE410 and KYSE150 cells were co-transfected with LCN2-HA/LOXL2-Flag, LCN2-HA/MMP9-Flag, or LOXL2-HA/MMP9-Flag plasmid pairs, or the corresponding vector plasmids. High expression of the target proteins was observed in KYSE410 and KYSE150 after transfection, accordingly (Supplementary Fig. S1E).
The interaction patterns of intracellular and extracellular LCN2/LOXL2/MMP9
The protein-protein interaction network (PPIN) results showed that MMP9 interacts with both LCN2 and LOXL2. To confirm this, the endogenous interactions between three proteins were detected by immunofluorescence (IF). It revealed that in the KYSE150 cell line, LCN2 and LOXL2, LCN2 and MMP9, and LOXL2 and MMP9 were all co-localized, with the Pearson correlation coefficients as 0.79, 0.92 and 0.91, respectively (Fig. 1A). Therefore, the ternary complex of LCN2/LOXL2/MMP9 might exist in esophageal cancer cells. To further confirm the presence of LCN2/LOXL2/MMP9 ternary complexes in ESCC cells, co-immunoprecipitation using whole cell lysates was performed after co-transfection of LCN2-HA/LOXL2-Flag, LCN2-HA/MMP9-Flag, or LOXL2-HA/MMP9-Flag.
Considering LCN2, LOXL2 and MMP9 are all secreted proteins, the culture supernatant after transfection was also collected and concentrated to detect their extracellular interactions. The results showed that both in KYSE410 and KYSE150 cells, protein-protein interaction between LCN2 and LOXL2, and between LCN2 and MMP9, occurred both intracellularly and extracellularly (Fig. 1B-C). Nevertheless, the protein-protein interactions between LOXL2 and MMP9 occurred only intracellularly (Fig. 1D). Next, we co-overexpressed LCN2-HA/LOXL2-HA/MMP9-Flag in KYSE410 cells and carried out co-immunoprecipitation using the whole cell lysates and culture supernatants. We found protein-protein interactions between MMP9 and LCN2 both intracellularly and extracellularly, whereas the protein-protein interaction between MMP9 and LOXL2 was identified only intracellularly (Fig. 1E). The schematic intracellular and extracellular protein-protein interaction pattern of LCN2/LOXL2/MMP9 is shown in Fig. 1F.
LCN2/LOXL2/MMP9 protein-protein interaction promotes migration and invasion of ESCC cells
Since LCN2, LOXL2 and MMP9 form a ternary complex in esophageal cancer cells, their biological function regarding the ternary complex remained to be explored. In the KYSE150 cell line, wound healing experiments showed that co-overexpression of LCN2/LOXL2, LCN2/MMP9, and LOXL2/MMP9 increased the migration of ESCC cells (Fig. 2A). Moreover, both transwell assays and invasion assays showed LCN2/LOXL2, LCN2/MMP9, LOXL2 /MMP9 enhanced the migration and invasion of esophageal cancer cells (Fig. 2B-C). Similar results were also obtained with the KYSE410 cell line (Supplementary Fig. S2A-C). Compared to the solely transfected LCN2 group, either co-transfection of LCN2/LOXL2 or co-transfection of all three significantly improved the migration and invasion of esophageal cancer cells (Supplementary Fig. S2D).
In addition to migration and invasion, we also investigated the functional role of the LCN2/LOXL2/MMP9 ternary complex in cell proliferation. Colony-forming assays showed that overexpression of LCN2/LOXL2, LCN2/MMP9, LOXL2/MMP9 enhanced cell colony formation in the KYSE150 cell line (Fig. 2D). Similar results were obtained with the KYSE410 cell line (Supplementary Fig. S2E). MTS assay results showed that proliferation of ESCC cells in the experimental group did not change significantly after their overexpression in either KYSE410 or KYSE150 cells (Supplementary Fig. S2F-G). In the KYSE150 cell line, flow cytometry showed no significant cell cycle change in the experimental group (Supplementary Fig. S2H).
To obtain a stably overexpressing cell line, the NeonTM Transfection System (Thermo) was used to electroporate the LCN2-HA/LOXL2-HA/MMP9-Flag (abbreviated as LLM) plasmids into the KYSE150 cell line, followed by two weeks of G418 selection. Western blotting showed that a cell line stably overexpressing all three proteins was successfully established (Fig. 2E). Consistently, wound healing experiments showed that LLM increased the migration of esophageal cancer cells (Fig. 2F). In order to study the effect of LLM on ESCC tumor progression in vivo, we separately injected the cells stably overexpressing the ternary LLM complex and control cells into the footpads of nude mice. Compared with the control group, LLM cells displayed enhanced tumor progression (Fig. 2G). The anatomy results showed that 4 out 6 nude mice overexpressing LLM developed tumor metastasis, where the tumor cells migrated from the footpad to the ankle, and the lymph nodes of 2 nude mice were swollen (Fig. 2H).
LCN2/ LOXL2 and MMP9/ LOXL2 interactions depend on the SRCR domains
To identify the specific domains for LOXL2/LCN2, and LOXL2/MMP9 protein-protein interaction in the ternary complex, two truncated LOXL2 plasmids were constructed, DLOXL2-HA (545-774aa) (lacking the N-terminal scavenger receptor cysteine-rich (SRCR) domains, denoted D4-HA), and DLOXL2-HA (1-544aa) (lacking the C-terminal amine oxidase domain, denoted D9-HA), respectively. First, these two plasmids were able to be expressed in KYSE150 cells (Fig. 3A). Immunofluorescence showed that in the KYSE150 cell line, D4-HA did not co-localize with either LCN2 or MMP9, with Pearson correlation coefficients of -0.27 and 0.21, respectively (Fig. 3B). Confocal Z-stack analysis was applied to scan different sections of transfected cells to confirm their co-localization (Supplementary Fig. S3). Further, co-immunoprecipitation results showed that, the protein-protein interactions between LOXL2 and either LCN2 or MMP9 disappeared when the SRCR domains of LOXL2 were deleted (Fig. 3C-D). These results suggested the protein-protein interactions for LCN2/LOXL2 and MMP9/LOXL2 both depended on the SRCR domains of LOXL2. Furthermore, the biological roles of the N-terminus and C-terminus of LOXL2 was investigated. Transwell and invasion assays showed that the two truncated plasmids (D4 and D9) could reduce the ability of ESCC cells to migrate and invade, as well as colonize, compared to full-length LOXL2 (Fig. 3E-G).
LCN2 inhibitor DFOM inhibits the migration and tumor growth of ESCC cells
Deferoxamine mesylate (DFOM) is an iron chelator that binds free ferric iron and is mainly used to treat diseases caused by iron overload (19). KYSE150 cells were treated with 0, 5, 10, 25, 50, 100 and 200 μM DFOM. After 48 h, an MTS assay was performed, and the IC50 was calculated to be 30.993 μM. Therefore, 30 μM DFOM was used for subsequent experiments. DFOM could decrease the expression level of LCN2 in KYSE150 cells (Fig. 4A). Wound healing experiments showed that LCN2 overexpression-mediated enhancement of ESCC cell migration could be reduced by DFOM treatment (Fig. 4B). Compared with the stable overexpression of LLM, these results show that the expression of LCN2 can be significantly reduced after DFOM treatment, indicating that DFOM may destroy the formation of the ternary complex LLM or reduce its expression (Fig. 4C). Compared with empty vector group, the migration of ESCC cells could be significantly promoted by LLM, which was decreased after DFOM treatment (Fig. 4D). Colony-forming assays found that LCN2 or LLM could promote the proliferation ability of single ESCC cells, which could be inhibited by DFOM treatment (Fig. 4E). In order to study the effect of LCN2 and the LLM ternary complex on tumor progression of ESCC in vivo, as well as the anti-tumor effect of DFOM in vivo, a xenograft model was applied to observe and evaluate. Compared with the control group, LCN2 promoted ESCC tumor growth in vivo. But after intraperitoneal administration of DFOM, it was found that both the tumor volume and weight were significantly reduced (Fig. 4F). Similarly, the LLM ternary complex promoted tumor progression in vivo compared to the vector group. Consistently, tumor growth was inhibited, and the tumor volume and weight were also reduced after DFOM treatment (Fig. 4G).
LCN2/LOXL2/MMP9 promote the degradation of ECM
The LCN2/LOXL2/MMP9 ternary complex can promote the migration and invasion of ESCC cells, but the specific molecular mechanism is still unclear. In the experimental groups that overexpressed LCN2/MMP9 or LOXL2/MMP9, the ability to degrade gelatin for MMP9 was elevated in zymography experiments (Fig. 5A). It was also observed that gelatin degradation at 130kD occurred for extracts of LCN2/MMP9 co-overexpressing cells, which was corresponding to a LCN2/MMP9 complex. In cells co-overexpressing LCN2/MMP9 or LOXL2/MMP9, gelatin degradation was also seen at around 200kD, which coincides with the predicted molecular weight of the LCN2/LOXL2/MMP9 ternary complex. We presumed that when co-overexpressing LCN2/MMP9 or LOXL2/MMP9, a LCN2/LOXL2/MMP9 ternary complex synergistically degrades the matrix, promoting the migration and invasion of ESCC cells (Fig. 5A).
Next, visualization of ECM degradation in living cells was carried out using fluorescent matrix degradation assays. In KYSE150 cells co-overexpressing LCN2/LOXL2, LCN2/MMP9 and LOXL2/MMP9, the ability to degrade fluorescent Matrigel was enhanced, as a significant black ghost was formed after degradation, whereas Matrigel degradation was barely observed in control cells (Fig. 5B). Three sets of co-overexpressing cells were scanned from top to bottom using the Z-stack function of the confocal microscope. The results confirmed that cells co-overexpressing LCN2/LOXL2, LCN2/MMP9 or LOXL2/MMP9 degraded fluorescent Matrigel around the cell spheroids (Supplementary Fig. S4).
The growth environment of tumor cells in vivo is three-dimensional. For this reason, we further characterized the ability of tumor cells to degrade ECM in a 3D cell co-culture model. Cells in the control group displayed a round and smooth morphological structure, except for the enlargement of the cell body itself. In the LCN2/LOXL2-, LCN2/MMP9- or LOXL2/MMP9-overexpressing groups, in addition to enlargement of the cell body, the ability of cells to degrade Matrigel was enhanced as more Matrigel surrounding the cells was digested. With the prolongation of culture time, elongation and branching of filopodia around the cells became clearly visible (Fig. 5C). In the stably overexpressing LCN2/LOXL2/MMP9 cell line, a similar result was obtained that LLM can significantly degrade Matrigel and promote the extension of filopodia (Fig. 5D).
MMP9 activity in the ternary complex was examined. The zymography results indicated that the ternary complex LLM could significantly enhance the enzymatic activity of MMP9 to degrade gelatin and form a distinct degradation band, while DFOM treatment could significantly weaken the enzymatic activity of MMP9 (Fig. 5E). The results of 3D cell co-culture found that after DFOM treatment of ESCC cells, the ability of the LLM complex to degrade Matrigel was reduced along with the number of filopodia (Fig. 5F).
Since the interaction of LCN2/LOXL2 and MMP9/LOXL2 depends on the SRCR domains of LOXL2, we further detected the importance of the SRCR domains. The formation of filopodia was observed in cells transfected with truncated LOXL2 and the full-length LOXL2 after 48 h (Supplementary Fig. S5). The number and extension length of filamentous pseudopods of cells transfected with full-length LOXL2 were the most prominent, compared to those in cells transfected with the catalytic domain (D4) (Supplementary Fig. S5). Minimal change was observed in cells transfected with the SRCR domain (D9), indicating that the catalytic domain of LOXL2 plays an important role in ECM degradation and filopodia formation (Supplementary Fig. S5).
LCN2/LOXL2/MMP9 induce cytoskeletal microfilament remodeling
Tumor cell migration and invasion usually involve rearrangement of the cellular microfilament system. In order to better observe changes in the actin cytoskeleton of ESCC cells, F-actin in KYSE410 cells was fluorescently labeled after the overexpression of target proteins. The results showed that stress fibers in control cells were arranged in regular bundles, while the stress fibers in the overexpression group were more disordered, suggesting that overexpression of LCN2, LOXL2 and MMP9 could promote cell migration by remodeling the cell microfilament cytoskeleton (Fig. 6A). Profilin 1 (PFN1) is a member of the profilin family of small actin-binding proteins that play an important role in actin dynamics by regulating actin polymerization in response to extracellular signals . To observe the actin arrangement, F-actin and profilin 1 were simultaneously fluorescence labeled after LCN2/LOXL2, LCN2/MMP9 and LOXL2/MMP9 were transfected. The microfilament skeleton in the three overexpressing groups was more disordered, compared with the control group (Fig. 6B). The protein level of profilin 1 was increased and accumulated in the cytoplasm and at the leading edge of the cell. These results indicated that LCN2/LOXL2/MMP9 promotes cell migration by enhancing profilin 1 expression and promoting microfilament rearrangement (Fig. 6B).
In the KYSE410 cell line, LCN2-HA/LOXL2-HA/MMP9-Flag was overexpressed, and the HA and Flag tags were fluorescently labeled. It could be observed that LCN2, LOXL2, and MMP9 were all successfully over-expressed. Compared with the empty vector group, the cytoskeleton of cells overexpressing LLM was disordered, and the number of filopodia was significantly increased (Fig. 6C). Next, we aimed to analyze whether DFOM treatment could rescue the cytoskeleton disorder. We found the fluorescence intensity of profilin1 increased in LLM-overexpressing cells, but was decreased after DFOM treatment and accompanied by restoration of the cytoskeleton organization to an orderly state (Fig. 6D). These results suggest that the LLM ternary complex may enhance the expression of profilin 1. To confirm this, western blotting showed the expression of profilin 1 was decreased after DFOM treatment (Fig. 6E).
LCN2/LOXL2/MMP9 activate the FAK/AKT/GSK3β signaling pathway
Next, we wanted to identify the signaling pathways involving the LLM. After LCN2, LOXL2 and MMP9 were overexpressed individually, the phosphorylation levels of NFkB, STAT3, and PTEN remained unchanged, while phosphorylation level of AKT increased, indicating this signaling pathway was activated (Fig. 7A). After co-overexpression of LCN2/LOXL2, LCN2/MMP9 and LOXL2/MMP9, the phosphorylation levels of NFkB, STAT3 and PTEN still remained unchanged, while the phosphorylation levels of FAK, AKT and GSK3β also increased, suggesting that LCN2/LOXL2/MMP9 protein-protein interaction promotes migration and invasion of ESCC cells by activating the FAK/AKT/GSK3β signaling pathway (Fig. 7B). Similar alternations were also observed in KYSE410 cells (Fig. 7C). On the other hand, co-overexpression of LCN2/LOXL2, LCN2/MMP9 or LOXL2/MMP9 induced no significant change the level of phosphorylated ERK (Supplementary Fig. S6).
For further confirmation, pathway activators or inhibitors were applied to confirm the involvement of these signaling pathways. Capivasertib (MCE, HY-15431), which increases phosphorylation of AKT at both Ser473 and Thr308 (21), was added to KYSE150 cells to mimic the activation by LCN2/LOXL2/MMP9 complex. Compared with the control group, AKT phosphorylation was enhanced by capivasertib, and the expression level of LCN2, LOXL2 and MMP9 were increased as well (Fig. 7D). Wortmannin is a pan-inhibitor of PI3K. When cells overexpressing LCN2/LOXL2/MMP9 were grown mixed with Matrigel in a 3D model to observe the changes of cell morphology, no obvious changes were observed in filopodia number and length except the enlarge of cell body itself. However, filopodia in the overexpression group began to form at 3 h after transfection. After 6 h, most cells in overexpressing group had formed filopodia that extended around the cell surface, thus promoting cell spreading, morphological change and cell migration. On the other hand, LCN2/LOXL2/MMP9-overexpressing cells treated with wortmannin had barely extended filopodia after 6 h, and only part of cells began to form the short filamentous pseudopods (Fig. 7E).
The SPOCK1 gene encodes a matricellular glycoprotein that belongs to a family of novel Ca2+-binding proteoglycans that promote cell migration in many tumors . Expression of SPOCK1 was clearly increased after LCN2/LOXL2/MMP9 overexpression, to reshape the ECM, then promote the migration and invasion of ESCC cells. Next, the PI3K inhibitor wortmannin was added to cells overexpressing LCN2/LOXL2/MMP9. Wortmannin treatment resulted in inhibition of SPOCK1 expression, suggesting that the ternary complex of LCN2/LOXL2/MMP9 activates the AKT signaling pathway to promote SPOCK1 expression (Fig. 7F). Interestingly, overexpression of SPOCK1 in KYSE150 cells enhanced phosphorylation of AKT, suggesting a positive regulation feed-back loop between SPOCK1 and p-AKT involved by LCN2/LOXL2/MMP9 (Fig. 7G).