Abstract

Expression of L1 cell adhesion molecule (L1CAM) is associated with poor prognosis in a variety of human carcinomas including breast, ovarian and pancreatic ductal adenocarcinoma (PDAC). Recently we reported that L1CAM induces sustained nuclear factor kappa B (NF-κB) activation by augmenting the autocrine production of interleukin 1 beta (IL-1β), a process dependent on interaction of L1CAM with integrins. In the present study, we demonstrate that transforming growth factor β1 (TGF-β1) treatment of breast carcinoma (MDA-MB231) and PDAC (BxPc3) cell lines induces an EMT (epithelial to mesenchymal transition)-like phenotype and leads to the expression of L1CAM. In MDA-MB231 cells, up-regulation of L1CAM augmented expression of IL-1β and NF-κB activation, which was reversed by depletion of L1CAM, L1CAM-binding membrane cytoskeleton linker protein ezrin, β1-integrin or focal adhesion kinase (FAK). Over-expression of L1CAM not only induced NF-κB activation but also mediated the phosphorylation of FAK and Src. Phosphorylation was not induced in cells expressing a mutant form of L1CAM (L1-RGE) devoid of the integrin-binding site. FAK- and Src-phosphorylation were inhibited by knock-down of various components of the integrin signalling pathway such as β1- and α5-integrins, integrin-linked kinase (ILK), FAK and the phosphoinositide 3-kinase (PI3K) subunit p110β. In summary, these results reveal that during EMT, L1CAM promotes IL-1β expression through a process dependent on integrin signalling and supports a motile and invasive tumour cell phenotype. We also identify important novel downstream effector molecules of the L1CAM–integrin signalling crosstalk that help to understand the molecular mechanisms underlying L1CAM-promoted tumour progression.

Introduction

Tumour cells show a high plasticity allowing them to respond and adapt to environmental influences ( 1 ). The pathological activation of the epithelial to mesenchymal transition (EMT) is a hallmark of aggressive tumours and has been implicated in the promotion of cancer cell invasion, metastasis and drug resistance ( 2–6 ). EMT is characterized by a change in cell morphology, loss of epithelial characteristics, e.g. E-cadherin expression, and the acquisition of mesenchymal markers, e.g. vimentin ( 7 , 8) . In vitro, EMT can be induced by different extracellular signals such as transforming growth factor β (TGF-β) ( 9 ), epidermal growth factor ( 10 ) or ligation of the Notch receptor ( 11 ). TGF-β has been described as one of the most potent EMT inducers for cultured cells ( 7 ). We have recently observed that the L1 cell adhesion molecule (L1CAM) is up-regulated in endometrial carcinoma (EC) and pancreatic ductal adenocarcinoma (PDAC) by TGF-β treatment ( 12 , 13) . In EC cells, the TGF-β1-induced up-regulation of L1CAM is accompanied by a decrease in E-cadherin expression, which is blocked by knock-down of the transcription factor SLUG ( 12 ). Similar results have been reported for PDAC cells ( 13 ). Thus, under appropriate conditions, L1CAM is regulated in an EMT-like fashion.

L1CAM is a 200–220kDa transmembrane glycoprotein of the immunoglobulin superfamily composed of six immunoglobulin-like domains and five fibronectin type III repeats followed by a transmembrane region and a highly conserved cytoplasmic tail ( 14 ). It can interact with itself (homophilic) and with a variety of heterophilic ligands such as integrins, CD24, neurocan, neuropilin-1 and other members of the neural cell adhesion family ( 15 ). In the nervous system, L1CAM promotes cell survival, migration and axon outgrowth ( 15–17 ). In many human cancers, L1CAM is constitutively over-expressed and its expression is generally associated with poor prognosis and metastases formation (for review, see refs 17 , 18 ).

Thus, the mechanisms of L1CAM signalling are of interest for the field of neurobiology and cancer research. It has been shown that L1CAM expression in neural and tumour cells activates the MAP kinase signalling pathway ( 19–22 ). Furthermore, L1CAM signals to the nucleus through a process termed regulated intramembrane proteolysis whereby the cytoplasmic part is sequentially cleaved by a disintegrin and metalloproteinase 10 and presenilins and can translocate into the nucleus ( 23 ). Recently, L1CAM over-expression in tumour cells has been shown to induce nuclear factor kappa B (NF-κB) activation via enhanced production of interleukin 1 beta (IL-1β) in an integrin-dependent manner ( 24 , 25) . The L1CAM-mediated NF-κB activation requires binding of the linker protein ezrin to the cytoplasmic portion of L1CAM and promotes liver metastasis formation ( 26 ).

In the present study, we address the role of TGF-β1-induced L1CAM expression in the promotion of IL-1β production and NF-κB activation. We also extensively analyse the role of integrin downstream signalling in the L1CAM-mediated NF-κB activation and induction of IL-1β. Additionally, we prove on a functional level that TGF-β-induced L1CAM expression correlates with enhanced cell motility and cell invasion.

Material and methods

Chemicals and antibodies

Antibodies to the ectodomain of L1CAM (monoclonal antibody L1-11A, a subclone of UJ127.11 and monoclonal antibody L1-9.3) were described ( 22 , 27) . The antibody to vimentin (Zymed, V9, Invitrogen, Eggenstein, Germany) has been described before ( 12 ). Antibodies against β1-integrin, α5-integrin, ILK, FAK, Src, PI3Kβ and the phosphorylated residues FAK-Y925, Src-Y416, Akt-S473, and p65-S536 were obtained from Cell Signaling (New England Biolabs, Frankfurt a.M., Germany). Antibody directed against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was obtained from Santa Cruz (Heidelberg, Germany). Other antibodies used were β-Actin (Sigma–Aldrich, Taufkirchen, Germany), PI3K-p110β-Y384 (Abcam, Cambridge, UK) and phosphorylated IκBα-S32 (Epitomics, Biomol, Hamburg, Germany). Recombinant IL-1β was obtained from R&D Systems (Frankfurt, Germany).

Cell culture

The human cell line PT45-P1 was originally obtained from Dr Kalthoff (University of Kiel, Kiel, Germany) ( 28 , 29) and retrovirally transduced with an empty vector (mock), full-length L1CAM (L1-FL) an L1CAM molecule with a mutation in the RGD sequence (L1-RGE) or an L1CAM molecule with a deletion of the cytoplasmic part (L1-1147) ( 23 , 24) . The Kalthoff lab recently determined that the PT45-P1 cells were mixed up with the breast cancer cell line MDA-MB231 ( 30 ). This was found out by DNA fingerprinting performed by DSMZ Braunschweig, Germany. We reconfirmed that our transduced cells were indeed MDA-MB231 cells (by testing the rare CD24 polymorphisms, rs3838646) ( 31 ) and renamed these cells MDA-MB231-L1CAM and MDA-MB231-mock. We received the authentic PDAC cell line PT45-P1 cells from Dr Kalthoff (University of Kiel) and transduced them with empty vector or L1CAM as described above (PT45-P1-mock, PT45-P1-L1CAM, respectively). The PDAC cell lines BxPc3 (L1CAM negative) and Panc-1 (L1CAM positive) were from ATCC-LGC (Wesel, Germany). All cell lines were cultivated in RPMI-1640 supplemented with 10% fetal calf serum at 37°C, 5% CO 2 and 100% humidity. For the induction of EMT, cells were cultivated in medium in the presence of HGF (5ng/ml) or TGF-β1 (10 ng/ml) for 10 days.

Biochemical analysis

Cells were seeded into 6-well plates and treated as indicated. For cell lysis, 100 µl 2x NuPage LDS sample buffer (Invitrogen, Karlsruhe, Germany) were added per well and the lysates were sonicated. To analyse protein phosphorylation, cells were grown to 70% confluence in 10cm plates. Lysis was performed with 100 µl PathScan Sandwich enzyme-linked immunosorbent assay (ELISA) Lysis Buffer (Cell Signaling) and lysates were centrifuged according to the manufacturer’s protocol. Protein concentration was determined by protein assay (BCA, Thermo Fisher Scientific, Schwerte, Germany). SDS-PAGE and transfer of separated proteins to immobilon membranes using semi-dry blotting was described before ( 32 ).

Flow cytometry

The staining of cells with monoclonal antibodies and PE- or Alexa488-conjugated secondary antibodies has been described ( 24 , 33) .

Small interfering RNA transfection

All cells were transfected with Interferin (Polyplus, Illkirch, France) according to manufacturer’s protocol. Small interfering RNA (siRNA) sequences are listed in the Supplementary Material, available at Carcinogenesis Online.

Quantitative real-time PCR

Quantitative real-time PCR (qRT-PCR) was performed as described before ( 23 ). Primers for qRT-PCR were designed using the IDT primer quest software and were produced by MWG (Ebersberg, Germany). β-Actin was used as an internal standard. The sequences of primers used are available on request. In brief, qRT-PCR measurements were done in triplicates for each experiment and each experiment was repeated at least thrice. Data analysis was performed using the delta-delta Ct method. Bar charts depict results of a single representative experiment including technical variability across triplicates. In order to test the regulation effect for each cell line based on independent biological replicates, we performed a two-sided paired t -test (delta Ct_target gene versus delta Ct_reference gene; delta Ct = Ct_target gene − Ct_reference gene) with pairing according to experiment. Technical triplicates per experiment were averaged. Statistical significances of a minimum of three independent biological replicates are depicted in Supplementary Table 1 , available at Carcinogenesis Online.

Luciferase assay

Cells (1×10 5 ) were seeded into 6-well plates, grown for 24h and then subjected to jetPEI transfection (Polyplus) using 1 µg pGL3-(κB)3-Luc (firefly luciferase; T.Hofmann, DKFZ, Heidelberg, Germany) or pGL3-SV40-Luc (firefly luciferase) with 0.02 µg pRLTK ( Renilla luciferase ). The assay was carried out as described previously ( 24 , 34) .

ELISA assays

Cells (1×10 6 ) were seeded into 10cm plates and were grown for 48h. Supernatants were concentrated by centrifugal ultrafiltration using Vivaspin concentrators (Sartorius Stedim, Göttingen, Germany). Levels of secreted human IL-1β were quantified using the Quantikine-HS human IL-1β immunoassay (R&D Systems). The ELISA was performed according to the manufacturer’s instructions. Measured IL-1β concentrations were normalized to protein concentration of whole cell extracts determined in parallel. The NF-κB-specific ELISA was carried out as described in detail before ( 35 ).

Transmigration and matrigel invasion assays

The transmigration assay has been described before ( 27 ). Each experiment was done in quadruplicates and the mean values ± SD are presented. Tumour cell invasion in vitro was determined in a double-filter assay as described ( 22 , 36) .

Statistical analysis

For the analysis of statistical significance the Student’s t- test was used. P -values in the figures are indicated as follows: *<0.05, **<0.01 and ***<0.001.

Results

TGF-β1-induced EMT is associated with the up-regulation of L1CAM in carcinoma cells

TGF-β is a major inducer of EMT during development, tissue morphogenesis and carcinogenesis ( 2–6 ). We have recently demonstrated that treatment of EC cells with TGF-β1 induces expression of L1CAM ( 12 ). To expand this observation to other cancer cell types, the breast cancer cell line MDA-MB231 and the PDAC cell line BxPc3 were treated with either TGF-β1 or hepatocyte growth factor (HGF) for 10 days and analysed for L1CAM expression. Only TGF-β1 induced L1CAM expression in both cell lines, as determined at the messenger RNA (mRNA) and protein levels ( Figure 1A –D).

Fig. 1.

Regulation of L1CAM expression during EMT. ( A and B ) MDA-MB231 and BxPc3 cells were treated with TGF-β1 or HGF and the regulation of L1CAM, E-cadherin, keratin 8, keratin 19, vimentin and SLUG was analysed by qRT-PCR. A representative experiment from n ≥ 3 independent experiments is shown. ( C and D ) Cell lysates from treated or non-treated cells were subjected to Western blot analysis using antibodies to L1CAM, vimentin and β-Actin. After incubation with the primary antibody, blots were incubated with the respective peroxidase-conjugated secondary antibody followed by enhanced chemiluminescence detection. Representative blots of n ≥ 3 experiments are shown. Note that E-cadherin was undetectable in MDA-MB231 cells.

Fig. 1.

Regulation of L1CAM expression during EMT. ( A and B ) MDA-MB231 and BxPc3 cells were treated with TGF-β1 or HGF and the regulation of L1CAM, E-cadherin, keratin 8, keratin 19, vimentin and SLUG was analysed by qRT-PCR. A representative experiment from n ≥ 3 independent experiments is shown. ( C and D ) Cell lysates from treated or non-treated cells were subjected to Western blot analysis using antibodies to L1CAM, vimentin and β-Actin. After incubation with the primary antibody, blots were incubated with the respective peroxidase-conjugated secondary antibody followed by enhanced chemiluminescence detection. Representative blots of n ≥ 3 experiments are shown. Note that E-cadherin was undetectable in MDA-MB231 cells.

As we did not observe remarkable morphological changes (data not shown), we assessed the effects of TGF-β1 on the expression of protein markers to confirm EMT induction in the treated cell lines. The epithelial markers E-cadherin, keratin 8 and keratin 19 were down-regulated in MDA-MB231 cells, whereas keratin 8 and keratin 19 were down-regulated in BxPc3 cells ( Figure 1A and 1B ). Although not normally expressed by BxPc3 cells the mesenchymal marker vimentin became clearly detectable after TGF-β1 treatment and its expression was increased in the vimentin-positive MDA-MB231 cells. We also detected an up-regulation of the EMT-related transcription factor SLUG in both cell lines ( Figure 1A and 1B ). Treatment of cells with HGF did not induce notable changes in gene expression ( Figure 1A –D). These results suggest that L1CAM induction is part of a functional TGF-β1-mediated cellular EMT programme.

EMT induction is accompanied by the up-regulation of IL-1β in carcinoma cells

Ectopic expression of L1CAM in MDA-MB231 cells induces IL-1β expression and NF-κB activation, which plays a major role in L1CAM functions ( 24 ). We next analysed whether L1CAM up-regulation mediated through a more physiological stimulus, such as TGF-β1, activated similar signalling mechanisms. We observed that TGF-β1-induced L1CAM expression in MDA-MB231 and BxPc3 cells promoted a strong increase in IL-1β mRNA levels and enhanced secretion of the protein into the medium ( Figure 2A and 2B ). In contrast, HGF treatment neither up-regulated IL-1β mRNA expression nor enhanced its secretion ( Figure 2A and 2B ). Induction of L1CAM in MDA-MB231 cells was also accompanied by increased NF-κB activation as measured by an NF-κB-specific ELISA ( Figure 2A , right panel). Although BxPc3 cells secreted enhanced levels of IL-1β after TGF-β1 stimulation, no increase in NF-κB activation was observed ( Figure 2B , right panel). As autocrine activation of NF-κB by IL-1β requires the presence of a signalling competent IL-1β receptor, we examined the responsiveness towards recombinant IL-1β. MDA-MB231, PT45-P1 and Panc-1 cells had a strong activation of NF-κB, the BxPc3 cells showed a weak responsiveness ( SupplementaryFigure 1A , available at Carcinogenesis Online). It should be noted that BxPc3 cells exhibit high endogenous secretion of IL-1β and NF-κB activation ( 37 , 38) , possibly accounting for their irresponsiveness to additional IL-1β. To confirm that the up-regulation of L1CAM in MDA-MB231 cells was instrumental for the increase in IL-1β levels, we depleted L1CAM by specific knock-down after TGF-β1-mediated EMT induction. Loss of L1CAM expression prevented IL-1β production ( Figure 3A ) and NF-κB activation ( Figure 3B ), supporting the hypothesis that TGF-β1-induced L1CAM promotes these signalling events during EMT.

Fig. 2.

TGF-β1 treatment augments IL-1β expression and NF-κB activity. ( A and B ) mRNA was isolated from TGF-β1 or HGF-treated MDA-MB231 (A) or BxPc3 (B) cells and cDNAs were analysed by qRT-PCR for IL-1β (left panel). A representative experiment from n ≥ 3 independent experiments is shown. IL-1β protein content was determined by an IL-1β-specific ELISA in supernatants of densely grown cells (middle panel). Pooled data from n = 3 experiments are shown. NF-κB activity was measured using a phospho-p65-specific ELISA. Pooled data from n ≥ 3 experiments are shown.

Fig. 2.

TGF-β1 treatment augments IL-1β expression and NF-κB activity. ( A and B ) mRNA was isolated from TGF-β1 or HGF-treated MDA-MB231 (A) or BxPc3 (B) cells and cDNAs were analysed by qRT-PCR for IL-1β (left panel). A representative experiment from n ≥ 3 independent experiments is shown. IL-1β protein content was determined by an IL-1β-specific ELISA in supernatants of densely grown cells (middle panel). Pooled data from n = 3 experiments are shown. NF-κB activity was measured using a phospho-p65-specific ELISA. Pooled data from n ≥ 3 experiments are shown.

Fig. 3.

IL-1β expression is L1CAM and integrin dependent. ( A ) Control or TGF-β1-treated MDA-MB231 cells were transfected with either control siRNA (GFP siRNA) or L1CAM siRNA. Knock-down efficiency and effects of L1CAM depletion on IL-1β gene expression were analysed by qRT-PCR. A representative experiment from n = 3 independent experiments is shown. ( B ) NF-κB activity of the transfected cells was determined by a luciferase reporter assay. Luciferase activity was determined in whole cell lysates and normalized to internal and SV40 controls. A representative experiment from n = 3 independent experiments is shown. ( C–E ) Control or TGF-β1-treated MDA-MB231 cells were transfected with either control siRNA (GFP siRNA or ctrA siRNA) or siRNAs to β1-integrin, FAK or ezrin. Knock-down efficiency and effects of the respective knock-downs on IL-1β gene expression were analysed by qRT-PCR. A representative experiment from n ≥ 3 independent experiments is shown.

Fig. 3.

IL-1β expression is L1CAM and integrin dependent. ( A ) Control or TGF-β1-treated MDA-MB231 cells were transfected with either control siRNA (GFP siRNA) or L1CAM siRNA. Knock-down efficiency and effects of L1CAM depletion on IL-1β gene expression were analysed by qRT-PCR. A representative experiment from n = 3 independent experiments is shown. ( B ) NF-κB activity of the transfected cells was determined by a luciferase reporter assay. Luciferase activity was determined in whole cell lysates and normalized to internal and SV40 controls. A representative experiment from n = 3 independent experiments is shown. ( C–E ) Control or TGF-β1-treated MDA-MB231 cells were transfected with either control siRNA (GFP siRNA or ctrA siRNA) or siRNAs to β1-integrin, FAK or ezrin. Knock-down efficiency and effects of the respective knock-downs on IL-1β gene expression were analysed by qRT-PCR. A representative experiment from n ≥ 3 independent experiments is shown.

Recently, binding of the membrane–actin cytoskeleton linker protein ezrin to L1CAM was found to be required for L1CAM-dependent NF-κB activity pointing to the importance of the L1CAM cytoplasmic domain in L1CAM signalling ( 26 ). In line with this study, we confirm the requirement of an intact cytoplasmic domain for L1CAM-dependent NF-κB activation. The L1-1147 mutant, which lacks the cytoplasmic tail, failed to promote IL-1β secretion and NF-κB activation when stably expressed in MDA-MB231 cells ( Supplementary Figure 1E , available at Carcinogenesis Online). We also demonstrate that knock-down of ezrin in TGF-β1-treated MDA-MB231 cells abolished the L1CAM-induced expression of IL-1β ( Figure 3E ). Thus, our data provide first evidence for a link between the role of ezrin in L1CAM-mediated NF-κB activation and L1CAM-dependent IL-1β induction, which is also supported by L1CAM–integrin interactions.

Integrin signalling mediates L1CAM-dependent IL-1β expression during EMT

Various reports have implicated a crosstalk of TGF-β and integrin signalling during EMT ( 39 , 40) . Using siRNA knock-downs of L1CAM, α5-integrin or integrin-linked kinase (ILK), we have recently shown that L1CAM–α5-integrin interactions are instrumental for the L1CAM-mediated IL-1β induction ( 24 ). To analyse whether integrin signalling is also relevant for IL-1β expression in TGF-β1-stimulated L1CAM expressing cells, we depleted the β1-integrin subunit (forms the α5β1 heterodimer) and the integrin-associated focal adhesion kinase (FAK) by siRNA-mediated knock-down ( Figure 3C and 3D ). Loss of either β1-integrin or FAK led to a reduction of IL-1β levels ( Figure 3C and 3D ), supporting the role of integrin signalling in the L1CAM-mediated IL-1β induction during EMT.

L1CAM-dependent integrin signalling is required for NF-κB activation

To dissect L1CAM-mediated activation of integrin downstream signalling in more detail, we used MDA-MB231 cells and the PDAC cell line PT45-P1 stably transduced with empty vector (mock) or L1CAM [( 24 ) and SupplementaryFigure 2A and B, available at Carcinogenesis Online]. L1CAM-transduced cells showed high L1CAM expression as detected by qRT-PCR and fluorescence-activated cell sorting analysis [( 24 ) and SupplementaryFigure 2A and B, available at Carcinogenesis Online]. Similar to MDA-MB231-L1CAM cells ( 24 ), PT45-P1-L1CAM cells had an increased IL-1β expression and NF-κB activation compared with mock cells ( SupplementaryFigure 2C and D, available at Carcinogenesis Online).

Integrin activation and ligand binding is followed by binding of intracellular proteins like ILK, FAK and Src, which are key regulators of integrin downstream signalling ( 41 , 42) . An early event in the integrin signalling pathway is the auto-phosphorylation of FAK-Y397 and Src-Y416 leading to their activation ( 41 ). We observed 2-fold increased phosphorylation of FAK at Y397 in L1CAM expressing MDA-MB231 cells, when compared with mock cells ( Figure 4A ). L1CAM expression further resulted in the increased phosphorylation of Src at Y416, which was accompanied by FAK phosphorylation at the Src-specific site Y925 ( Figure 4A ). In addition to the increased activation of the integrin signalling components, an enhanced phosphorylation of the NF-κB subunit p65 at S536 was observed in L1CAM expressing cells ( Figure 4A ). As integrin activation induces signalling pathways, which are also regulated by TGF-β1, including the PI3-kinase-Akt pathway ( 40 , 43) , we analysed the phosphorylation of PI3K subunit p110β and Akt-S473. The phosphorylation of both signalling components was increased in MDA-MB231-L1CAM cells ( Figure 4A ). Similar activation of FAK-Y397 and Akt-S473 was also observed in PT45-P1 cells expressing L1CAM ( Supplementary Figure 2E , available at Carcinogenesis Online).

Fig. 4.

L1CAM expression induces integrin signalling. ( A ) Lysates from mock-, L1CAM- or L1-RGE-transduced MDA-MB231 cells were subjected to Western blot analysis using antibodies to the indicated phosphorylation epitopes. GAPDH was used as loading control. ( B ) Effects of L1CAM or L1-RGE expression in MDA-MB231 cells on the depicted NF-κB target genes were analysed by qRT-PCR. ( C ) Panc-1 cells were transfected with either control siRNA (GFP siRNA) or L1CAM siRNA. Lysates of the transfected cells were subjected to western blot analysis using antibodies to the indicated phosphorylation epitopes. GAPDH was used as loading control. ( D ) Effects of L1CAM depletion on the depicted NF-κB target genes in Panc-1 cells were analysed by qRT-PCR. Note that the quantification of band intensities in (A) and (C) are indicated relative to the GAPDH loading control. Representative data from n = 3 independent experiments are shown.

Fig. 4.

L1CAM expression induces integrin signalling. ( A ) Lysates from mock-, L1CAM- or L1-RGE-transduced MDA-MB231 cells were subjected to Western blot analysis using antibodies to the indicated phosphorylation epitopes. GAPDH was used as loading control. ( B ) Effects of L1CAM or L1-RGE expression in MDA-MB231 cells on the depicted NF-κB target genes were analysed by qRT-PCR. ( C ) Panc-1 cells were transfected with either control siRNA (GFP siRNA) or L1CAM siRNA. Lysates of the transfected cells were subjected to western blot analysis using antibodies to the indicated phosphorylation epitopes. GAPDH was used as loading control. ( D ) Effects of L1CAM depletion on the depicted NF-κB target genes in Panc-1 cells were analysed by qRT-PCR. Note that the quantification of band intensities in (A) and (C) are indicated relative to the GAPDH loading control. Representative data from n = 3 independent experiments are shown.

We have previously demonstrated that the L1CAM-RGE mutant (L1-RGE), which is deficient in integrin binding, is incapable of inducing L1CAM-mediated IL-1β expression and NF-κB activation when expressed in MDA-MB231 cells ( 24 ). L1-RGE failed to activate the integrin signalling pathway as phosphorylation of the tested kinases was impaired compared with L1CAM-transduced cells ( Figure 4A ).

Supporting the role of L1CAM–integrin signalling in NF-κB activation, we detected increased expression of the NF-κB target genes IκBα, IL-6, IL-8 and IL-13 in MDA-MB231-L1CAM cells ( Figure 4B ), whereas no significant changes in gene expression were observed in L1-RGE expressing cells when compared with mock transfected cells ( Figure 4B ). Likewise, in PT45-P1-L1CAM cells, we detected strong induction of IκBα, IL-6 and IL-8 mRNA expression ( Supplementary Figure 2F , available at Carcinogenesis Online).

These data demonstrate for the first time that L1CAM–integrin interactions mediated through the L1CAM-RGD-binding site activate classical integrin downstream signalling, ultimately leading to NF-κB activation.

L1CAM and integrin signalling in Panc-1 cells

To expand these observations to a cell line endogenously expressing L1CAM, we analysed the effect of L1CAM depletion on integrin-associated kinases in Panc-1 cells. We have previously shown that IL-1β expression and constitutive NF-κB activity depend on L1CAM in these cells ( 24 ). Here we observed that L1CAM depletion also led to reduced phosphorylation of FAK-Y397, FAK-Y925, Src-Y416, PI3Kβ-Y384, Akt-S473 and p65-S276 ( Figure 4C ). qRT-PCR analysis of mRNA derived from L1CAM-depleted cells showed that the expression of the above mentioned NF-κB target genes IκBα, IL-6, IL-8 and IL-13 was impaired, as well ( Figure 4D ).

To validate the role of integrin signalling in Panc-1 cells, we employed siRNA-mediated knock-down of various components of the integrin signalling pathway including α5-integrin, β1-integrin, ILK, FAK and the PI3K subunit p110β. Knock-down efficiency was determined by qRT-PCR and western blot analysis ( Supplementary Figure 3 , available at Carcinogenesis Online) and the phosphorylation status of FAK–Src and Akt was analysed ( Supplementary Figure 4 , available at Carcinogenesis Online).

We observed that the elimination of each component resulted in a diminished IL-1β expression and NF-κB activation ( Figure 5A –F) and strongly reduced transcription of IL-8, a well-known NF-κB target gene (data not shown). Taken together, these data confirm that L1CAM-mediated integrin signalling leads to activation of FAK and Src culminating in the increased expression of IL-1β, a prerequisite for constitutive NF-κB activity.

Fig. 5.

NF-κB activation is mediated via integrin signalling. ( A–F ) Panc-1 cells were depleted for L1CAM ( A ), β1-integrin ( B ), α5-integrin ( C ), ILK ( D ), PI3K subunit p110β ( E ) or FAK ( F ). Analysis of IL-1β expression was performed using qRT-PCR. NF-κB activation was analysed by luciferase reporter assay. Representative data from n = 3 independent experiments are shown.

Fig. 5.

NF-κB activation is mediated via integrin signalling. ( A–F ) Panc-1 cells were depleted for L1CAM ( A ), β1-integrin ( B ), α5-integrin ( C ), ILK ( D ), PI3K subunit p110β ( E ) or FAK ( F ). Analysis of IL-1β expression was performed using qRT-PCR. NF-κB activation was analysed by luciferase reporter assay. Representative data from n = 3 independent experiments are shown.

L1CAM-triggered NF-κB activity augments cell migration and invasion

EMT has been associated with an increased migration and invasiveness of tumour cells ( 2 , 3) . Previous work has shown that L1CAM triggers cell migration and invasion ( 20 , 27 , 44) . In our setting, L1CAM over-expression, as well as TGF-β treatment, increased the cell migration on type I collagen in a transwell migration assay ( Figure 6A ) and we observed similar effects on cell invasion in matrigel invasion assays ( Figure 6B ). To investigate whether L1CAM expression accounted for TGF-β1-induced cell invasion, we used siRNA knock-downs to deplete L1CAM from TGF-β1-treated MDA-MB231 cells. Loss of L1CAM significantly reduced cell invasion ( Figure 6C ), which was also blocked by the specific NF-κB inhibitor parthenolide ( Figure 6D ).

Fig. 6.

L1CAM expression augments migration and invasion. ( A ) The indicated cells were analysed for haptotactic cell migration. Collagen type I was used as substrate and coated onto the lower side of transwell chambers. After 16h the transmigrated cells retained in the substrate were stained with crystal violet solution. The eluted dye was measured at 595nm in an ELISA reader. Each determination was done in quadruplicates. The migration of mock cells was set to 100%. Pooled data from n = 3 independent experiments are shown. ( B ) Analysis of matrigel invasion. Indicated cells were seeded into a 4-well plate and were allowed to invade into matrigel using a double filter assay. After 22h the invaded cells adhering to the top of the second membrane were counted after staining with 4′,6-diamidino-2-phenylindole. Data are given as % invaded cells after correction for cell proliferation. ( C ) The indicated cells were depleted for L1CAM by siRNA-mediated knock-down and invasion was analysed as described above. A representative experiment from n = 3 independent experiments is shown. ( D ) The described invasion assay was performed in the presence of 1 µM parthenolide or dimethyl sulfoxide as control. A representative experiment from n = 3 independent experiments is shown. ( E ) Schematic illustration of L1CAM-mediated integrin signalling. L1CAM triggers integrin signalling through the phosphorylation and activation of the downstream components FAK, Src and PI3K. Activation of integrin signalling leads to elevated levels of IL-1β expression on the mRNA and protein level. Secreted IL-1β binds in an autocrine fashion to the IL-1β receptor. Triggering of the receptor leads to constitutive NF-κB activation. Note that IL-1β is both a target gene for NF-κB and an activator of NF-κB. It is therefore possible that NF-κB activation occurs first leading to increased IL-1β production.

Fig. 6.

L1CAM expression augments migration and invasion. ( A ) The indicated cells were analysed for haptotactic cell migration. Collagen type I was used as substrate and coated onto the lower side of transwell chambers. After 16h the transmigrated cells retained in the substrate were stained with crystal violet solution. The eluted dye was measured at 595nm in an ELISA reader. Each determination was done in quadruplicates. The migration of mock cells was set to 100%. Pooled data from n = 3 independent experiments are shown. ( B ) Analysis of matrigel invasion. Indicated cells were seeded into a 4-well plate and were allowed to invade into matrigel using a double filter assay. After 22h the invaded cells adhering to the top of the second membrane were counted after staining with 4′,6-diamidino-2-phenylindole. Data are given as % invaded cells after correction for cell proliferation. ( C ) The indicated cells were depleted for L1CAM by siRNA-mediated knock-down and invasion was analysed as described above. A representative experiment from n = 3 independent experiments is shown. ( D ) The described invasion assay was performed in the presence of 1 µM parthenolide or dimethyl sulfoxide as control. A representative experiment from n = 3 independent experiments is shown. ( E ) Schematic illustration of L1CAM-mediated integrin signalling. L1CAM triggers integrin signalling through the phosphorylation and activation of the downstream components FAK, Src and PI3K. Activation of integrin signalling leads to elevated levels of IL-1β expression on the mRNA and protein level. Secreted IL-1β binds in an autocrine fashion to the IL-1β receptor. Triggering of the receptor leads to constitutive NF-κB activation. Note that IL-1β is both a target gene for NF-κB and an activator of NF-κB. It is therefore possible that NF-κB activation occurs first leading to increased IL-1β production.

These data confirm and extend the previous findings that L1CAM expression drives migration and invasion, suggesting that cooperation of L1CAM with integrins induces NF-κB activity, which in turn promotes a motile and invasive cancer cell phenotype.

Discussion

Although L1CAM is widely expressed in neural cells and over-expressed in a variety of human carcinomas, L1CAM-dependent signalling capabilities are still incompletely understood and mainly described in non-tumour cells. Herein we demonstrate that L1CAM expression is up-regulated in carcinoma cells by treatment with the EMT inducer TGF-β1. Concomitant with and dependent on L1CAM expression, we observe augmented production of IL-1β, activation of NF-κB and enhanced expression of NF-κB target genes in several cancer cell lines. We show that L1CAM-mediated NF-κB signalling functions through the activation of the integrin-FAK-Src-Akt signalling pathway, dependent on an intact RGD–integrin binding sequence in the L1CAM molecule. Thus, our results unravel for the first time an L1CAM-dependent signalling pathway in which L1CAM acts as a ligand for integrins thereby promoting NF-κB activation. This pathway (termed L1CAM reverse signalling) should be considered as an independent pathway, in addition to the previously described signalling of L1CAM via nuclear translocation of the C-terminal proteolytic fragment (termed L1CAM forward signalling) ( 25 ).

In a previous study, we have shown that L1CAM is induced by TGF-β1 treatment in EC and PDAC cells ( 12 , 13) . Here we expand our observations and demonstrate that TGF-β1 treatment of other cell lines including MDA-MB231 and BxPc3 induces an EMT-like phenotype with loss of epithelial markers (reducing keratins 8 and 19, and E-cadherin in the case of MDA-MB231) and enhanced expression of the mesenchymal marker vimentin and the transcription factor SLUG. Importantly, L1CAM expression parallels the up-regulation of vimentin and SLUG. This is in line with recent immunohistochemical analysis supporting the hypothesis that L1CAM is up-regulated by cancer cells as part of the EMT process. L1CAM expression on EC and lung cancer tissue sections was confined to areas with loss in E-cadherin expression ( 12 , 45) .

Recently, we provided evidence that ectopic expression of L1CAM in MDA-MB231 cells is intimately linked to NF-κB activation via the induction of IL-1β, a classical inducer of NF-κB transcriptional activity ( 24 ). In this study, we extend these findings to the PDAC cell line PT45-P1 and show increased IL-1β production and NF-κB activation in response to L1CAM expression. Including PT45-P1 cells into the study was of significance due to the mix-up of cell lines as described in the Materials and methods section. Importantly, not only the ectopic expression of L1CAM in cancer cell lines from different tumour types but also the physiologic induction of L1CAM by TGF-β1 in MDA-MB231 cells and the PDAC cell line BxPc3 induced L1CAM-dependent IL-1β production. Collectively, these data support a more general role of L1CAM in the regulation of IL-1β expression independent of the tumour type.

Our previous work in MDA-MB231 cells demonstrated that integrin signalling is instrumental for the induction of IL-1β expression and NF-κB activation, and that the knock-down of α5-integrin or the integrin-associated adapter molecule ILK abolished IL-1β induction and subsequent NF-κB activity ( 24 , 46) . Herein we show that also TGF-β1-induced IL-1β expression was dependent on the expression of L1CAM, β1-integrin and FAK. In Panc-1 cells, depletion of L1CAM, as well as of β1-integrin, ILK, PI3Kβ and FAK, also prevented IL-1β production and NF-κB activity. In addition to IL-1β, we also observed down-regulation of other common NF-κB target genes, i.e. IκBα, IL-6, IL-8 or IL-13. These data suggest that the interaction of L1CAM with integrins is essential to stimulate NF-κB activity and NF-κB-dependent gene regulation.

L1CAM is known to bind integrins such as α5β1- or αv-integrins and several reports have shown a link between the expression of L1CAM and enhanced FAK activation ( 47–49 ). The kinases FAK, Src and PI3Kβ were described as classical components of integrin signalling ( 42 , 50) . Using the MDA-MB231 L1CAM over-expression model, we unravelled an L1CAM-dependent signalling pathway in which the L1CAM molecule acts as an integrin ligand inducing the phosphorylation of the downstream signalling components FAK, Src, PI3K and Akt, leading to NF-κB activation. In contrast, the kinases were not phosphorylated in cells expressing the mutant L1-RGE form devoid of integrin-binding capacity, underlining the importance of direct L1CAM–integrin interaction via the RGD-binding site.

Tumour cells often express both L1CAM and L1CAM-binding integrins such as α5β1- or αv-integrin simultaneously. We favour the interpretation that L1CAM–integrin interactions occur in trans (in opposing membranes). However, we cannot formally rule out a binding in cis (i.e. in the same membrane) (see model Figure 6D ).

In addition to the role of L1CAM interactions through the extracellular domain a recent study pointed to the importance of the L1CAM cytoplasmic domain in L1CAM-induced NF-κB activitation. The authors show that ezrin, an adapter molecule that can bind to the cytoplasmic part of L1CAM ( 51 ) and other adhesion molecules, is required for L1CAM-mediated NF-κB activity in colon cancer cells ( 26 ). Our study demonstrates a role of ezrin in the L1CAM-mediated induction of IL-1β in TGF-β1-treated MDA-MB231 cells. We show that ezrin knock-down prevents IL-1β production and NF-κB activation. Similar results were obtained in MDA-MB231-L1CAM over-expressing cells (H.Kiefel, unpublished results). Taken together our data provide a link between ezrin and the L1CAM–integrin signalling platform in the induction of NF-κB signalling during EMT.

EMT and NF-κB activation promote metastasis of breast cancer cells ( 52 ). L1CAM expression enhances cell motility, increases matrigel invasion, augments tumour growth in NOD–Scid mice and metastases formation (20–22,26,27,44,53–55). L1CAM-driven metastasis of colon cancer cells to the liver is inhibited by over-expression of a NF-κB superrepressor ( 26 ). In line with this study, we found that either L1CAM over-expression or TGF-β1 treatment augmented migration and invasion of MDA-MB231 cells. The enhanced invasion was blocked by knock-down of L1CAM or by treatment of cells with the NF-κB inhibitor parthenolide. Our results suggest that L1CAM-augmented NF-κB activity is a driving force in promoting cell invasion and motility that are essential for tumour metastasis.

In previous studies, we have investigated the signalling mechanisms of L1CAM in human tumours that were summarized in a recent review article ( 25 ). The results in this report add a more detailed investigation of the downstream elements of L1CAM–integrin signalling (see model Figure 6E ). These novel insights will contribute to our understanding of the role of L1CAM in tumour progression.

Supplementary material

Supplementary Table 1 and Figures 1–4 can be found at http://carcin.oxfordjournals.org/

Funding

Deutsche Krebshilfe Project Nr 108739

Deutsche Forschungsgemeinschaft (SE-1831/2-1 to S.S.); EU-FP6 framework programme OVCAD (PE-14034 to P.A.).

Acknowledgements

We thank Veronika Jahndel, Angelika Schmidt and Thomas Mock for the primer allocation of NF-κB target genes. We thank Thomas Hielscher for the assistance with statistical analyses.

Conflict of Interest Statement: None declared.

References

1.
Grivennikov
S.I.
et al
2010
Immunity, inflammation, and cancer.
Cell
 
140
883
899
2.
Christiansen
J.J.
et al
2006
Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis.
Cancer Res.
 
66
8319
8326
3.
Polyak
K.
et al
2009
Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits.
Nat. Rev. Cancer
 
9
265
273
4.
Cheng
G.Z.
et al
2007
Twist transcriptionally up-regulates AKT2 in breast cancer cells leading to increased migration, invasion, and resistance to paclitaxel.
Cancer Res.
 
67
1979
1987
5.
Thiery
J.P.
2002
Epithelial-mesenchymal transitions in tumour progression.
Nat. Rev. Cancer
 
2
442
454
6.
Arumugam
T.
et al
2009
Epithelial to mesenchymal transition contributes to drug resistance in pancreatic cancer.
Cancer Res.
 
69
5820
5828
7.
Thiery
J.P.
et al
2006
Complex networks orchestrate epithelial-mesenchymal transitions.
Nat. Rev. Mol. Cell Biol.
 
7
131
142
8.
Gavert
N.
et al
2008
Epithelial-mesenchymal transition and the invasive potential of tumors.
Trends Mol. Med.
 
14
199
209
9.
Zavadil
J.
et al
2005
TGF-beta and epithelial-to-mesenchymal transitions.
Oncogene
 
24
5764
5774
10.
Lo
H.W.
et al
2007
Epidermal growth factor receptor cooperates with signal transducer and activator of transcription 3 to induce epithelial-mesenchymal transition in cancer cells via up-regulation of TWIST gene expression.
Cancer Res.
 
67
9066
9076
11.
Leong
K.G.
et al
2007
Jagged1-mediated Notch activation induces epithelial-to-mesenchymal transition through Slug-induced repression of E-cadherin.
J. Exp. Med.
 
204
2935
2948
12.
Huszar
M.
et al
2010
Up-regulation of L1CAM is linked to loss of hormone receptors and E-cadherin in aggressive subtypes of endometrial carcinomas.
J. Pathol.
 
220
551
561
13.
Geismann
C.
et al
2009
Up-regulation of L1CAM in pancreatic duct cells is transforming growth factor beta1- and slug-dependent: role in malignant transformation of pancreatic cancer.
Cancer Res.
 
69
4517
4526
14.
Moos
M.
et al
1988
Neural adhesion molecule L1 as a member of the immunoglobulin superfamily with binding domains similar to fibronectin.
Nature
 
334
701
703
15.
Brummendorf
T.
et al
1998
Neural cell recognition molecule L1: from cell biology to human hereditary brain malformations.
Curr. Opin. Neurobiol.
 
8
87
97
16.
Schachner
M.
1997
Neural recognition molecules and synaptic plasticity.
Curr. Opin. Cell Biol.
 
9
627
634
17.
Schafer
M.K.
et al
2010
L1CAM malfunction in the nervous system and human carcinomas.
Cell Mol. Life Sci.
 
67
2425
2437
18.
Gavert
N.
et al
2008
L1-CAM in cancerous tissues.
Expert Opin. Biol. Ther.
 
8
1749
1757
19.
Schaefer
A.W.
et al
1999
Activation of the MAPK signal cascade by the neural cell adhesion molecule L1 requires L1 internalization.
J. Biol. Chem.
 
274
37965
37973
20.
Silletti
S.
et al
2004
Extracellular signal-regulated kinase (ERK)-dependent gene expression contributes to L1 cell adhesion molecule-dependent motility and invasion.
J. Biol. Chem.
 
279
28880
28888
21.
Gavert
N.
et al
2005
L1, a novel target of beta-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers.
J. Cell Biol.
 
168
633
642
22.
Gast
D.
et al
2008
The cytoplasmic part of L1-CAM controls growth and gene expression in human tumors that is reversed by therapeutic antibodies.
Oncogene
 
27
1281
1289
23.
Riedle
S.
et al
2009
Nuclear translocation and signalling of L1-CAM in human carcinoma cells requires ADAM10 and presenilin/gamma-secretase activity.
Biochem. J .
 
420
391
402
24.
Kiefel
H.
et al
2010
L1CAM-integrin interaction induces constitutive NF-kappaB activation in pancreatic adenocarcinoma cells by enhancing IL-1beta expression.
Oncogene
 
29
4766
4778
25.
Kiefel
H.
et al
2011
Linking L1CAM-mediated signaling to NF-kappaB activation.
Trends Mol. Med.
 
17
178
187
26.
Gavert
N.
et al
2010
Nuclear factor-kappaB signaling and ezrin are essential for L1-mediated metastasis of colon cancer cells.
J. Cell Sci.
 
123
2135
2143
27.
Mechtersheimer
S.
et al
2001
Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins.
J. Cell Biol.
 
155
661
673
28.
Kalthoff
H.
et al
1993
p53 and K-RAS alterations in pancreatic epithelial cell lesions.
Oncogene
 
8
289
298
29.
Sebens Muerkoster
S.
et al
2007
Drug-induced expression of the cellular adhesion molecule L1CAM confers anti-apoptotic protection and chemoresistance in pancreatic ductal adenocarcinoma cells.
Oncogene
 
26
2759
2768
30.
Lemke
J.
et al
2010
TRAIL signaling is mediated by DR4 in pancreatic tumor cells despite the expression of functional DR5.
J. Mol. Med.
 
88
729
740
31.
Marme
F.
et al
2012
CD24 Ala57Val polymorphism predicts pathologic complete response to sequential anthracycline- and taxane-based neoadjuvant chemotherapy for primary breast cancer.
Breast Cancer Res. Treat.
 
132
819
831
32.
Gutwein
P.
et al
2000
Role of Src kinases in the ADAM-mediated release of L1 adhesion molecule from human tumor cells.
J. Biol. Chem.
 
275
15490
15497
33.
Issa
Y.
et al
2009
Enhanced L1CAM expression on pancreatic tumor endothelium mediates selective tumor cell transmigration.
J. Mol. Med.
 
87
99
112
34.
Pfeifer
M.
et al
2010
L1CAM expression in endometrial carcinomas is regulated by usage of two different promoter regions.
BMC Mol. Biol.
 
11
64
35.
Bondong
B.
et al
2011
Prognostic significance of L1CAM in ovarian cancer and its role in constitutive NFκB activation
Ann Oncol.
  2012;
23
(7)
:
1795
802
.
36.
Brunner
G.
et al
1998
Sulfated glycosaminoglycans enhance tumor cell invasion in vitro by stimulating plasminogen activation.
Exp. Cell Res.
 
239
301
310
37.
Arlt
A.
et al
2003
Role of NF-kappaB and Akt/PI3K in the resistance of pancreatic carcinoma cell lines against gemcitabine-induced cell death.
Oncogene
 
22
3243
3251
38.
Muerkoster
S.
et al
2005
Increased expression of the E3-ubiquitin ligase receptor subunit betaTRCP1 relates to constitutive nuclear factor-kappaB activation and chemoresistance in pancreatic carcinoma cells.
Cancer Res.
 
65
1316
1324
39.
Bhowmick
N.A.
et al
2001
Integrin beta 1 signaling is necessary for transforming growth factor-beta activation of p38MAPK and epithelial plasticity.
J. Biol. Chem.
 
276
46707
46713
40.
Mamuya
F.A.
et al
2012
aV integrins and TGF-β-induced EMT: a circle of regulation.
J. Cell Mol. Med.
 
16
445
455
41.
Guo
W.
et al
2004
Integrin signalling during tumour progression.
Nat. Rev. Mol. Cell Biol.
 
5
816
826
42.
Legate
K.R.
et al
2006
ILK, PINCH and parvin: the tIPP of integrin signalling.
Nat. Rev. Mol. Cell Biol.
 
7
20
31
43.
Margadant
C.
et al
2010
Integrin-TGF-beta crosstalk in fibrosis, cancer and wound healing.
EMBO Rep.
 
11
97
105
44.
Gast
D.
et al
2005
L1 augments cell migration and tumor growth but not beta3 integrin expression in ovarian carcinomas.
Int. J. Cancer
 
115
658
665
45.
Tischler
V.
et al
2011
L1CAM protein expression is associated with poor prognosis in non-small cell lung cancer.
Mol. Cancer
 
10
127
46.
Gast
D.
et al
2008
The RGD integrin binding site in human L1-CAM is important for nuclear signaling.
Exp. Cell Res.
 
314
2411
2418
47.
Stoeck
A.
et al
2007
L1-CAM in a membrane-bound or soluble form augments protection from apoptosis in ovarian carcinoma cells.
Gynecol. Oncol.
 
104
461
469
48.
Min
J.K.
et al
2010
L1 cell adhesion molecule is a novel therapeutic target in intrahepatic cholangiocarcinoma.
Clin. Cancer Res.
 
16
3571
3580
49.
Yang
M.
et al
2011
L1 stimulation of human glioma cell motility correlates with FAK activation.
J. Neurooncol.
 
105
27
44
50.
Guan
J.L.
2010
Integrin signaling through FAK in the regulation of mammary stem cells and breast cancer.
IUBMB Life
 
62
268
276
51.
Cheng
L.
et al
2005
L1-mediated branching is regulated by two ezrin-radixin-moesin (ERM)-binding sites, the RSLE region and a novel juxtamembrane ERM-binding region.
J. Neurosci.
 
25
395
403
52.
Huber
M.A.
et al
2004
NF-kappaB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression.
J. Clin. Invest.
 
114
569
581
53.
Gavert
N.
et al
2007
Expression of L1-CAM and ADAM10 in human colon cancer cells induces metastasis.
Cancer Res.
 
67
7703
7712
54.
Meier
F.
et al
2006
The adhesion molecule L1 (CD171) promotes melanoma progression.
Int. J. Cancer
 
119
549
555
55.
Thelen
K.
et al
2002
The neural cell adhesion molecule L1 potentiates integrin-dependent cell migration to extracellular matrix proteins.
J. Neurosci.
 
22
4918
4931

Abbreviations:

    Abbreviations:
  • EC

    endometrial carcinoma

  • ELISA

    enzyme-linked immunosorbent assay

  • EMT

    epithelial to mesenchymal transition

  • FAK

    focal adhesion kinase

  • GAPDH

    glyceraldehyde 3-phosphate dehydrogenase

  • HGF

    hepatocyte growth factor

  • IL-1β

    interleukin 1 beta

  • L1CAM

    L1 cell adhesion molecule

  • mRNA

    messenger RNA

  • NF-κB

    nuclear factor kappa B

  • PDAC

    pancreatic ductal adenocarcinoma

  • qRT-PCR

    quantitative real-time PCR

  • siRNA

    small interfering RNA;

  • TGF-β1

    transforming growth factor β1