A key axis of the N-WASP signaling pathway promotes distant metastasis in pancreatic cancer: LOXL2-FAK-N-WASP

doi:10.21203/rs.3.rs-1992553/v1

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A key axis of the N-WASP signaling pathway promotes distant metastasis in pancreatic cancer: LOXL2-FAK-N-WASP

https://doi.org/10.21203/rs.3.rs-1992553/v1

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Background

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid malignancies. A specific mechanism of its metastasis is not established. In our present study, we investigated whether Neural Wiskott-Aldrich Syndrome Protein (N-WASP) plays a role in distant metastasis of PDAC.

Methods

Pancreatic cancer cell lines MIA PaCa-2, PANC-1, AsPC-1, and BxPC-3 were used for in vitro and in vivo study. To evaluate the endogenous expression level of N-WASP, we purified the whole RNA and protein to perform the qPCR, RT-PCR and Western blot. And we confirmed the motility and invasiveness and the RNA-seq assays. By using of pancreatic cancer cell lines, orthotopic mouse model of pancreatic cancer was established.

Results

We found that N-WASP is markedly expressed in clinical patients with PDAC. Through the analysis of clinical patient samples, N-WASP positive group had a much more distant metastatic-pattern than N-WASP negative group. Moreover, it was turned out that N-WASP is a novel mediator of epithelial–mesenchymal transition (EMT) via gene expression profile studies. In addition, knockdown of N-WASP in pancreatic cancer cells had significantly inhibited cell invasion, migration, and EMT. We also observed that the lysyl oxidase-like 2 (LOXL2) and focal adhesion kinase (FAK) are positively associated with the N-WASP-mediated response, thereby modulating EMT and invadopodia. Both N-WASP and LOXL2 depletion significantly reduced the incidence of liver and lung metastatic lesions in orthotopic mouse models of pancreatic cancer.

Conclusions

These results clarify a new role for N-WASP signaling associated with LOXL2 in EMT and invadopodia that regulates intercellular communication in tumor cells to promote pancreatic cancer metastasis. These findings may aid in the development of therapeutic strategies against pancreatic cancer.

Pancreatic cancer

Neural Wiskott-Aldrich Syndrome Protein (N-WASP) signaling

Metastasis

Epithelial–mesenchymal transition(EMT)

Lysyl oxidase-like 2 (LOXL2)

Pancreatic ductal adenocarcinoma (PDAC) is known as a fatal disease. Despite advanced early screening and diagnosis, the overall 5-year survival rate remains unchanged. Even after surgery, early recurrence rate, local recurrence, and distant metastasis tend to be high in pancreatic cancer. Tumor metastasis is a multistep process wherein tumor cells disseminate from their primary site and form secondary tumors at a distant site. Metastasis occurs through a series of steps such as local invasion, intravasation, transport, extravasation, and colonization. Epithelial–mesenchymal transition (EMT), a developmental program, is important to promote metastasis in epithelium-derived carcinoma [1-6].

EMT transcription factors can also induce the formation of specialized subcellular structures called, “invadopodia” which invades the local extracellular matrix (ECM). Invadopodia play a key role in metastasis by local invasion [1, 7-11]. EMT not only allows carcinoma cells to dissociate from each other but also provides them the ability to degrade ECM for single-cell invasion to initiate the metastatic cascade. The critical initial steps in pancreatic cancer metastasis involve the detachment and invasion of tumor cells into the surrounding tissues which require changes to the adhesive and migratory properties of the tumor cells. It can be achieved partly through cell polarization and the extension of actin-rich membrane structures in the direction of movements such as filopodia, lamellipodia, or invadopodia found in invasive cancer cells [9, 12-15]. Recent studies suggest that Focal Adhesion Kinase (FAK) is involved in cancer progression through signaling by integrins and other cell surface receptors and plays an important role in the regulation of cell migration. The formation of membrane protrusions, which are crucial for cell motility, is controlled by the rearrangement of the actin cytoskeleton. FAK, which is crucial for FA turnover, inhibits ECM proteolysis. The specific role of FAK in the regulation of invadopodia formation and function in cancer cells [16-21].

Neural Wiskott-Aldrich Syndrome Protein (N-WASP) is a ubiquitously expressed member of the WASP family. This protein family is involved in changing cell morphology, such as invadopodium formation, growth, and correlation with certain cancer phenotypes. A high level of N-WASP expression positively correlates with progression and/or poor outcomes in lung, pancreatic, cervical cancer, hepatocellular, and esophageal squamous cell carcinoma [22-26]. However, the process of EMT-induced invadopodia in pancreatic cancer has not been elucidated yet. Hence, N-WASP could be highlighted as a potential therapeutic target related to the control of cancer progression.

Recently, we reported that lysyl oxidase-like 2 (LOXL2) is important for regulating EMT in pancreatic cancer [27]. However, the exact mechanism underlying LOXL2-mediated distant metastasis in pancreatic cancer has not been fully characterized. Therefore, in this present study, we investigated whether N-WASP expression is associated with LOXL2 via examining the development of pancreatic cancer using in vitro and in vivo models. We found that N-WASP mediates cancer progression by linking LOXL2 signaling to FAK signaling. Our findings indicate that LOXL2-induced N-WASP is a key regulator for tumor metastasis through invadopodia. These findings may aid in the development of therapeutic strategies against pancreatic cancer.

Patients

From June 2002 to December 2012, 81 patients underwent radical curative resection for pancreatic cancer at Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea. Among these patients, three were excluded due to poorly preserved tissue samples, incomplete clinicopathologic data, or loss to follow-up. Hence, 81 patients were retrospectively reviewed. Patients were followed every 3 months during the first 12 months, and then every 6 months after the first year. The study protocol was approved by the Institutional Review Board at Gangnam Severance Hospital, Yonsei University of Korea (3-2014-0153) and Institutional Animal Care and Use Committee (2019-0104) and complied with the Declaration of Helsinki. Informed consent was obtained from all participants.

Cell culture

MIA PaCa-2, PANC-1, BxPC-3, and AsPC-1 cells were obtained from American Type Culture Collection (ATCC) and cultured according to the ATCC guidelines. Cells were cultured in DMEM or RPMI (Biowest) supplemented with 10% fetal bovine serum (Biowest) and 1% antibiotic-anti mycotic (Gibco). Cells were incubated in a humidified atmosphere with 5% CO₂at 37℃.

siRNA interference

For gene knockdown, the following small interfering RNAs (siRNA) were purchased from Bioneer (Daejeon, KR). siLOXL2 (Bioneer, Cat.No 4017-1, sense : 5’-AGAUUCCGGAAAGCGUACA-3’/ antisense 5’-UGUACGCUUUCCGGAAUCU-3’)

si N-WASP (Bioneer, Cat.No 8976-1, sense: 5’-GUGCAUUAAUGGAAGUGAU-3’, antisense: 5’-AUCACUUCCAUUAAUGCAC-3’). Transfection was conducted using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturers’ instructions. Cells were harvested and processed 48–72 h post-transfection.

Antibodies and western blot

To prepare the lysates, frozen pancreatic cancer tissues and cells were washed with ice-cold phosphate buffer solution (PBS) and lysed in RIPA lysis buffer. Protein was separated by SDS-PAGE and transferred on nitrocellulose membranes, blocked in 5% skim milk, and incubated with respective primary antibodies (1:1000 dilution). Anti N-WASP, phospho-N-WASP, LOXL2 (Abcam), anti-vimentin and anti-snail (Cell Signaling), N-cadherin, E-cadherin (BD Bioscience), β-actin (Santa Cruz Biotechnology), and γ-tubulin (Sigma-Aldrich) were used. The membranes were washed twice with Tris-buffered saline and Tween 20 (TBST), followed by incubation with HRP-conjugated secondary antibody (1:7,000 dilution, Cell Signaling Technology) in TBST with 3% skim milk. Bound antibody was probed using the ECL solution (Bio-Rad). Chemiluminescent signals were captured using X-ray films (AGFA). All experiments were performed in triplicate.

RNA isolation and qPCR

Cells and tissues were collected, and RNA was isolated using TRIZOL Reagent® (Sigma-Aldrich) according to the manufacturer’s instructions. Of the total isolated RNA, 0.2 µg was analyzed by reverse transcriptase PCR using one-step RT-PCR kit (Intron, KR). The first-strand cDNA was synthesized with 1 µg of RNA as a template, using the RT-qPCR cDNA Synthesis Kit (Intron, KR), according to manufacturer’s instructions. RT-qPCR was performed using SYBR qPCR reaction mix (Applied Biosystems). The primer sequences used in this study are listed in Supplementary Table 1. The results are presented as a relative mRNA expression level calculated with the 2−ΔΔCT method, using Gapdh as a reference gene.

Migration and invasion assays

For scratch wound migration assay, transfected cells were plated at 1.2–1.4×10⁴on a 24-well plate. Wound scratches were made 24 h after plating. Images of migrated cells were taken every 24 h using a microscope. For invasion assays, 8-µm pore size wells in a Transwell system (Corning Inc.) were coated for 1 h with Matrigel (1:50, Corning) at room temperature. Transfected cells (2×10⁴)were seeded on the apical side of the Transwell chamber (24-well insert) in serum-free media and growth media was added to the basal compartment. The cells were allowed to invade for 24 h. The remaining cells on the top of the chamber were gently scraped off using wetted cotton swabs. The cells that had invaded the basal side were fixed in methanol for 10 min, stained with 0.2% crystal violet, and then washed multiple times with 3’DW. The migration and invasion assays were performed in triplicate and repeated three times independently.

Immunocytochemistry

Transfected cells were fixed with 3.7% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 30 min. Cells were washed with PBS thrice and incubated with 1% bovine serum albumin for 1 h at room temperature. Cells were incubated with primary antibodies (LOXL2, N-WASP, ARP2, phalloidin iFlour-488; Abcam) overnight at 4℃ (1:100 to 1:1000), and then, the cells were incubated at room temperature with Alexa Fluor 488 or 555 conjugated secondary antibodies (1:1000). FluoroShield mounting medium with DAPI (Abcam) was used stain and mount cells for 5 min. Immunostained cells were observed under a Carl Zeiss LSM780 confocal microscope.

Invadopodia formation assay

FITC-gelatin invadopodia assays were performed as per the manufacturer’s procedure (QCM™ Gelatin Invadopodia Assay, Millipore). Briefly, 8-well chamber slides were coated with poly-L-lysine for 20 min at room temperature, washed with PBS, fixed with glutaraldehyde for 15 min, and washed with DPBS three times. After washing, a gelatin mixture (1:5=Fluorescein gelatin:unlabeled gelatin) was applied to each well and incubated at room temperature for 10 min. Substrates disinfected with 70% ethanol in sterile water were incubated at room temperature for 30 min and protected from light. To eliminate residual free aldehydes, growth media were added to each well, incubated at room temperature for 30 min, and protected from light. Cells were plated in wells at 20–80% confluence and incubated for 24 h. After incubation time, slides were fixed with 3.7% formaldehyde in DPBS for 30 min at room temperature. Blocking or permeabilization buffer for fluorescent staining was treated for 1 h at room temperature. Slides were incubated in TRITC-phalloidin and DAPI in fluorescent staining buffer for 1 h. The chamber was removed and the slide was covered with mounting media. Imaging of invadopodia and ECM degradation was performed using fluorescence confocal microscopy (Carl Zeiss 780).

Knockdown stable cell lines

Five different short hairpin ribonucleic acid (shRNA) N-WASP sequences in a lentiviral vector (pLKO.1-Puro) and controlled shRNA were purchased from Sigma-Aldrich. For the production of lentiviruses and transfection to HEK293 cells, Lipofectamine 3000 (Invitrogen) was used. PANC-1 cells were infected with the collected and purified viral particles; cells were stably selected and maintained with 2 μg/ml puromycin treatment. The shRNA sequence that sufficiently knocked down N-WASP expression was GCACAACTTAAAGACAGAGAA. (TRCN0000123061, Sigma-Aldrich)

Immunoprecipitation

To analyze protein binding, cell lysates were incubated with anti-FAK or anti-ARP2 antibodies overnight at 4℃, and immune complexes were pulled down with protein A/G magnetic beads (EMD Millipore, USA). The interacted proteins were eluted in a denaturing SDS sample buffer and loaded on SDS-PAGE and transferred to a membrane. The membrane was blocked and incubated with indicated primary antibodies and stained using the ECL solution.

Pancreatic cancer orthotopic model

All animal studies were conducted with an approved protocol proposal from the animal ethics committee of Yonsei University College of Medicine (approval #2019-0104). BALB/c nude mice (age, 6 weeks, female) were purchased from Orient Bio. The pancreas was surgically exposed through an abdominal excision under anesthesia with an intraperitoneal injection (i.p.) of Alfaxan (25 mg/kg). Human pancreatic cancer cells were inoculated directly into the pancreas of the mice using a 30 G needle (BD Biosciences). PANC-1 cells (1 × 10⁶) were mixed with serum-free DMEM and Matrigel (1:1) and injected at a volume of 50 μl. To prevent cancer cells from leaking, the excision was closed after covering the injection site with the surge cell. After the operation, the mice were warmed and monitored until conscious and then placed in HEPA-filtered cages with feed and water. After 12 weeks, the mice were sacrificed and examined for tumor spreading through macroscopic and microscopic observations using hematoxylin and eosin (H&E) staining. The pancreas, spleen, liver, and lung were examined using the H&E section.

Immunohistochemical (IHC) staining

Serial sections (5 µm) of each block were adhered to poly-L-lysine-covered slides and incubated at 62 ˚C for 60 min. After deparaffinization and rehydration, the sections were heated in a 10-mM citrate buffer (pH 6.0) for 15 min and stained with an antibody. Normal pancreas tissue within the block was used as positive controls. IHC staining was categorized as negative, “1+”, “2+”, or “3+” in high-power fields (200×) according to the staining intensity. Positivity was assigned for the scores “2+” and “3+”. The staining intensity was assessed by two pathologists who were blind to the clinical outcomes.

Statistical methods

For the statistical analysis, the unpaired t-test and ANOVA were performed. The differences were considered statistically significant at p < 0.05—*; p ≤ 0.01—**. The tests were performed using GraphPad Prism version 8.01 (GraphPad Software).

N-WASP is highly expressed in pancreatic cancer cell lines and tissues

To elucidate the effects of N-WASP and its underlying molecular mechanisms, N-WASP expression was analyzed in a series of pancreatic cancer cell lines (MIA PaCa-2, PANC-1, AsPC-1, and BxPC-3). N-WASP was detected in all pancreatic cell lines (Fig. 1A). We investigated N-WASP expression in PDAC samples from 81 patients. In PDAC tissue, N-WASP overexpression was observed, whereas the normal pancreatic tissue had low expression of N-WASP assessed by IHC staining, western blotting, and RT-qPCR (Fig. 1B, 1C, 1D, 1E).

Clinical characteristics and patterns of recurrence according to N-WASP status

Patient characteristics, including expression of N-WASP, were evaluated using IHC staining described in Table 1. There was no significant difference in T stage, N stage, tumor stage, cell differentiation, perineural invasion, and lymphovascular invasion among patient groups based on the expression of N-WASP. N-WASP positive group showed much more distant metastatic- pattern (p= 0.004) than N-WASP negative group. (Table 2)

Table 1. Clinical characteristics of total patients according to N-WASP expression

	N-WASP negative (n=56)	N-WASP positive (n=25)	p value
Age	63.66±10.15	64.24±9.56	0.925
Sex
Men	26(46.4%)	14(56%)	0.426
Women	30(53.6%)	11(44%)
T stage(8th)
T1	9(16.1%)	4(16.0%)	0.414
T2	36(64.3%)	17(68%)
T3	11(19.6%)	3(12%)
Unknown	0(0%)	1(4%)
N stage(8th)
N0	19(33.9%)	6(24%)	0.645
N1	28(50%)	15(60%)
N2	9(16.1%)	4(16%)
Stage(8th)
IA	5(9.1%)	1(4%)	0.561
IB	8(14.5%)	2(8%)
IIA	6(10.9%)	2(8%)
IIB	27(49.1%)	15(60%)
III	9(16.4%)	4(16%)
Unknown	0(0%)	1(4%)
Perineural invasion
Positive	39(69.6%)	20(80%)	0.622
Negative	14(25%)	4(16%)
Unknown	3(5.4%)	1(4%)
Lymphovascular invasion
Positive	22(39.3%)	15(60%)	0.223
Negative	31(55.4%)	9(36%)
Unknown	3(5.4%)	1(4%)
Cell Differentiation
Well differentiation	11(19.6%)	2(8%)	0.168
Moderate differentiation	39(69.6%)	16(64%)
Poorly differentiation	5(8.9%)	5(20%)
Undifferentiation	1(1.8%)	2(8%)

Abbreviation. N-WASP, Neural Wiskott-Aldrich Syndrome Protein ; 8^th , The eighth edition American Joint Committee on Cancer staging system.

Table 2. Recurrence pattern of total patients between N-WASP negative patients and positive patients

N-WASP negative

(n=46)

N-WASP positive

(n=17)

p value

Local metastasis

27(58.7%)

3(17.6%)

0.004

Distant metastasis

19(41.3%)

14(82.4%)

Abbreviation. N-WASP, Neural Wiskott-Aldrich Syndrome Protein

N-WASP knockdown inhibits EMT in PDAC and reduces motility and invasiveness in pancreatic cancer cells

To evaluate whether N-WASP affects pancreatic cancer cells, N-WASP knockdown was applied in PANC-1 and AsPC-1 cells using siRNA. As shown in Fig. 2A, N-WASP knocked down cells exhibited significant increase in the expression of E-cadherin, whereas significant reduction in the expression of snail and vimentin. Moreover, the wound healing assay results revealed that the wound width was significantly reduced in the N-WASP-knockdown cells (Fig. 2B and F). The results demonstrated that the knockdown of N-WASP suppressed cell migration. Furthermore, transwell assay was performed to monitor whether N-WASP could affect pancreatic cancer cell migration and invasion. The results revealed nearly 70% reduction in cell migration and 50% reduction in cell invasion in N-WASP siRNA-transfected PANC-1 and AsPC-1 cells (Fig. 2C and E). These results suggest that inhibition of N-WASP suppresses PDAC progression.

Identification of potential target genes of N-WASP in pancreatic cancer

To investigate the mechanism of N-WASP in promoting PDAC progression, we firstly performed the RNA-seq assay to evaluate target gene expression changes triggered by N-WASP knocked down in PANC-1 cells. In RNA-seq, genes that showed different expression were identified after N-WASP knockdown. Also, several genes showed more than two-fold difference in expression compared to shControl cells (Fig. 3A and B). We further confirmed the potential pathway by GSEA pre-ranked tool analysis including all genes. GSEA revealed that N-WASP enrichment was associated with genes in “HALLMARK EMT” and “HALLMARK Cell migration” (Fig. 3C). Then, the gene ontology (GO) analysis associated differentially expressed genes with cell migration and EMT (Fig. 3D and E). Among the lower expressed genes of shN-WASP, many renowned genes associated with EMT and cell migration (e.g., GPC4, CCL5, CCL2, CTHRC1, CX3CL1, ICAM1, SPARC, BMP2, SNAI3, WNT11) are included. Some of those gene expressions were verified using qRT-PCR after the knockdown of N-WASP in PANC-1 cells (Fig. 3F).

FAK-dependent phosphorylation of N-WASP is regulated by LOXL2

Our previous studies showed that the FAK/LOXL2 axis is important in regulating EMT and metastasis in PDAC. To clarify whether LOXL2 affects N-WASP, we first evaluated the correlation of FAK-dependent phosphorylation of N-WASP by LOXL2. FAK-knockdown cells exhibited a significant reduction in the expression of phosphorylated N-WASP (Fig. 4A). LOXL2-knockdown cells exhibited a significant reduction in the expression of phosphorylated FAK (p-FAK) and phosphorylated N-WASP (p-N-WASP) in PANC-1 cells. We also analyzed AsPC-1 cells in which LOXL2 was overexpressed. LOXL2-overexpressed cells showed an increase in the expression of p-FAK and p-N-WASP in the PANC-1 cell (Fig. 4B). As shown in Fig. 4C, knockdown of LOXL2 expression decreased the efficiency of the interaction between N-WASP and FAK. Based on these findings, the FAK/N-WASP axis was significantly associated with LOXL2 expression in pancreatic cancer cells.

LOXL2 influences the actin filament regulation and invadopodia formation ability of N-WASP

Invadopodia formation is essential for invasion and metastasis. Emerging evidence revealed a critical role for cortactin in invadopodia formation as well as in promoting cell motility and invasion (Fig. 4D and 4E). Furthermore, knockdown of LOXL2 expression decreased the efficiency of the interaction between N-WASP and ARP2 (Fig. 4F and 4G). These results suggest that inhibition of the N-WASP/FAK/LOXL2 axis could reduce the progression of PDAC. To determine the effects of LOXL2 on invadopodia formation and investigate the expression of F-actin in PANC-1 cells regarding knockdown of LOXL2, the changes in cell morphology and invadopodia were recorded via immunofluorescence assay.

Regulation of N-WASP-LOXL2 axis affects invasiveness and expression of EMT markers in pancreatic cancer cells

To further explore the detailed molecular mechanisms by which the N-WASP-LOXL2 axis inhibits EMT, we used siRNAs to knock down N-WASP and LOXL2. The results were verified through western blot by comparing groups that are knocked down in each or both. A group of both N-WASP and LOXL2 knockdown significantly reduced EMT markers compared to each of the gene knockdown group. (N-cadherin, Vimentin, Snail; Fig. 5A). Silencing of N-WASP and LOXL2 significantly repressed migration and invasion in PANC-1 cells (Fig. 5B). Consistently, wound healing test showed that N-WASP and LOXL2 knockdown inhibits the wound healing ability (Fig. 5C). These data suggest that the N-WASP-LOXL2 axis promotes pancreatic cancer cell migration, invasion, and morphology change.

Inhibition of N-wasp reduces metastasis of pancreatic cancer to the liver and lungs

To confirm whether the LOXL2-N-WASP impacts the tumorigenesis of pancreatic cancer in vivo, PANC-1 cells transfected with shN-WASP or control vector were injected into the pancreas of nude mice. On the 84^th day following the orthotopic modeling, the body weight of the shN-WASP group was remarkably higher than that in the PANC-1 group. Similarly, the average tumor weight was lower in the shN-WASP group (Fig. 5D). Both liver and lung metastasis were lower in the shN-WASP group than in the PANC-1 group. The silencing of N-WASP can repress the pancreatic cancer progression in vivo, suggesting that N-WASP performs a substantial function in pancreatic cancer metastasis and proliferation (Fig. 5D and E). In microscopic features of the primary tumor in an orthotopic model of PANC-1 group, cancer cells expressed LOXL2 and N-WASP, confirmed by immunohistochemical staining for each primary antibody (Fig. 5G(A)). The expression of LOXL2 in the shN-WASP group was similar to that in the PANC-1 group, whereas N-WASP was decreased in the shN-WASP group (Fig. 5G(B)). The expression of LOXL2 was decreased and N-WASP was similar or slightly decreased in shLOXL2 group (Fig. 5G[C]; HE, hematoxylin and eosin; x200)

In this study, we demonstrated that the signaling pathway through the LOXL2-N-WASP axis contributes to tumor invasion and metastasis in pancreatic cancer through an integrated in vitro, in vivo, and clinical investigation. Our results showed that N-WASP is overexpressed in both pancreatic cancer lines and human pancreatic cancer tissues. Moreover, high expression of N-WASP was associated with increased distant metastasis and poor prognosis in vivo and clinical data. There are five known members of the WASP family: WAVE1-3, N-WASP, and WASP. Among these, N-WASP and WAVE-1 are important for invadopodia formation and ECM degradation, respectively. Thus, there are distinct pathways that activate branched actin assembly for invadopodia formation [28-36]. Invadopodia are subcellular structures that are selectively invasive as compared to non-invasive cancer cells and are involved in a pivotal process in cancer invasion [7, 13, 37].

We showed that N-WASP promotes proliferation and migration in pancreatic cancer cells. N-WASP increased colony formation in vitro and distant metastasis in vivo. Deletion of N-WASP inhibited cell proliferation and migration. Also, a high level of N-WASP expression was correlated with a high level of distant metastasis potential. The results of the mouse study showed that N-WASP was essential for invasion and metastasis in pancreatic cancer. We further investigated the relationship of N-WASP expression with clinicopathologic features by immunohistochemistry analysis of 81 pancreatic cancer tissues. N-WASP expression seemed to correlate with distant metastasis after surgery.

Our previous study showed that the expression of LOXL2 was higher in human pancreatic cancer than in normal pancreas. Also, the higher expression of LOXL2 pancreatic cancer displayed significantly greater potency in distant metastasis [27].

Invadopodia and podosomes share many common functional and molecular characteristics, including dependence on Src kinase and branched actin assembly, as well as common molecular components, including focal adhesion proteins, integrins, and proteases. Many molecules that localize to focal adhesions are found in invadopodia or podosomes, including cytoskeletal proteins such as alpha-actinin, VASP, and zyxin, integrin linkers such as vinculin, and talin, and signaling molecules such as FAK, paxillin, p130Cas, and ERK [38-41].

Moreover, positive modulation of the FAK and SRC pathways by LOXL2 has been reported, which contributes to cell migratory behavior. Recently, Baker et al. showed that LOXL2 activated fibroblasts through integrin-mediated FAK activation in tumor cell invasion and metastasis [42]. LOXL2 plays an integral role in EMT promotion and invasiveness of pancreatic cancer cells. Our in vitro study indicated that LOXL2 activates EMT-like processes in pancreatic cell lines that are associated with invasive and migratory properties. Moreover, LOXL2 positively contributes to the activation of FAK/SRC and influences the expression levels of CDH1, Snail, and L1CAM, which are all related to EMT and invasiveness of pancreatic tumor cells.

Therefore, we analyzed the axis leading to LOXL2-FAK-N-WASP. Specifically, to prove the effect of N-WASP on distant metastasis, an orthotopic mouse model was constructed and showed significant results. It is important to elucidate the role and mechanism of N-WASP in pancreatic cancer, specifically, its role in the metastasis process. We identified the signaling mechanism of N-WASP through RNA transcriptome sequencing and related gene analysis.

In conclusion, we showed that N-WASP has an important role in expression of genes associated with cell migration and EMT. These results suggest that N-WASP affects the metastasis of cancer cells. This study proved a correlation between the expression of N-WASP in pancreatic cancer and distant metastasis. The findings support emerging evidence that the LOXL2-N-WASP axis is crucial in regulating metastasis in pancreatic cancer.

Acknowledgments

We thank Ji Hae Nam, pathologist in Gangnam Severance Hospital for providing assistance and So Young Jun for assisting collecting patient samples in Gangnam Severance Hospital.

Authors' contributions

HSK and YSL performed study design, data collection, formal analysis, and experiments and writing of original draft and prepared tables and figures 1-5. JSP and SMD performed study design and investigation and review and editing. HJK, DEL, HWK, MJK carried out the experiments prepared figures 1-5. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The study protocol was approved by the Institutional Review Board at Gangnam Severance Hospital, Yonsei University of Korea (3-2014-0153) and complied with the Declaration of Helsinki. Informed consent was obtained from all participants.

Consent for publication

Informed consent was obtained from all participants.

Availability of data and materials

The datasets generated and/or analysed during the current study are available from the

corresponding author on reasonable request.

Competing interests

The authors declare no conflict of interest

Funding information

This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korean government(MSIP) (2016R1C1B102207).

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A key axis of the N-WASP signaling pathway promotes distant metastasis in pancreatic cancer: LOXL2-FAK-N-WASP

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