Nardilysin and ADAM proteases promote gastric cancer cell growth by activating intrinsic cytokine signalling via enhanced ectodomain shedding of TNF-α

Nardilysin (NRDc), a metalloendopeptidase of the M16 family, promotes ectodomain shedding of the precursor forms of various growth factors and cytokines by enhancing the protease activities of ADAM proteins. Here, we show the growth-promoting role of NRDc in gastric cancer cells. Analyses of clinical samples demonstrated that NRDc protein expression was frequently elevated both in the serum and cancer epithelium of gastric cancer patients. After NRDc knockdown, tumour cell growth was suppressed both in vitro and in xenograft experiments. In gastric cancer cells, NRDc promotes shedding of pro-tumour necrosis factor-alpha (pro-TNF-α), which stimulates expression of NF-κB-regulated multiple cytokines such as interleukin (IL)-6. In turn, IL-6 activates STAT3, leading to transcriptional upregulation of downstream growth-related genes. Gene silencing of ADAM17 or ADAM10, representative ADAM proteases, phenocopied the changes in cytokine expression and cell growth induced by NRDc knockdown. Our results demonstrate that gastric cancer cell growth is maintained by autonomous TNF-α–NF-κB and IL-6–STAT3 signalling, and that NRDc and ADAM proteases turn on these signalling cascades by stimulating ectodomain shedding of TNF-α.


Figure S1
Immunohistochemical analysis of NRDc expression in gastric cancer tissues. Additional images of gastric cancer specimens immunostained with anti-NRDc antibody are shown. Lower panels are magnified photographs of the boxed areas in the upper panels. Left panels are images of another region of the case 2 specimen (See Fig 1B). Scale bars, 100 μm. Right panel, cell lysates were prepared from TMK-1 cells without using protease inhibitors. Cell lysates were incubated with the increasing doses of TAPI-1 at 4 °C for 1 h, followed by the TACE activity assay. (C) Left panel, TMK cells were transfected with control or ADAM17 siRNA. At 72 h after tranfection, cell lysates were subjected to Western blotting. Asterisk, non-specific band. Right panel, TACE activity assay was performed using TMK-1 cell lysates transfected with control or ADMA17 siRNA. (D) The MTS assay was performed after TMK-1 cells were left untreated or treated with the indicated concentrations of TAPI-1 for 30 h.

Sandwich ELISA for serum
To establish a sandwich ELISA system, all combinations of the 7 monoclonal antibodies for NRDc were tested, and the optimum combination of clone #231 for coating and #304 for detection was determined. For measuring serum NRDc concentrations, an automated analyzer for the chemiluminescent enzyme immunoassay, SphereLight 180 (Olympus, Tokyo, Japan), was utilized according to the manufacturer's protocol. After optimization, human serum NRDc could be measured with high sensitivity and specificity, with a detectable limit of 50 pg/ml.

Immunohistochemistry
Human gastric cancer or mouse xenograft tumor tissues were fixed in 4% PFA for 24 h, transferred to PBS, and processed for embedding in paraffin. For immunostaining, 5 µm sections were incubated with the indicated primary antibodies (anti-NRDc mouse monoclonal antibody (#102, established in our laboratory), 1:1000; anti-phospho-STAT3 rabbit monoclonal antibody, 1:50) at room temperature for 2 h or at 4°C overnight. After rinsing with 5% washing solution, the specimens were reacted with biotinylated secondary antibody at room temperature for 1-2 h and incubated with the avidin-biotin-peroxidase complex (Vectastain ABC kit, Vector Laboratories, Burlingame, CA) at room temperature for 30 min.
Then, color was developed with diaminobenzidine solution (Dako, Glostrup, Denmark), followed by staining with hematoxylin.

Cell culture and preparation of CM
TMK-1, MKN-1, and MKN-45 human gastric cancer cell lines were described previously (Yokozaki, 2000). These cells were cultured in RPMI 1640 medium (Invitrogen/GIBCO) supplemented with 10% fetal bovine serum (growth medium). To prepare CM, confluent monolayers of the control or stable NRDc-KD TMK-1 cells were incubated in RPMI 1640 medium supplemented with 0.1% bovine serum albumin for 48 h. The cultured media were collected and centrifuged at 3,000 rpm at 4°C for 10 min, and the supernatant was harvested.

qRT-PCR
Total RNA was isolated from the cultured cells using TRIzol reagent (Invitrogen) or RNeasy mini kit (Qiagen, Valencia, CA). cDNA generated by reverse transcription was subjected to real-time PCR assay using LightCycler 480 and FastStart Universal SYBR Green Master (Roche Applied Sciences, Indianapolis, IN) according to the manufacturer's protocols. mRNA levels of tested genes were normalized to 18S rRNA or GAPDH levels using a delta Ct method. Quantification of both full-length (FL) and splice variant (SV) forms of IL-6R mRNA was performed as previously reported (Nakanishi et al, 2004). Used forward and reverse primers are described in Table SI of Supporting information.

Western blotting
Cells were lysed with ice-cold 1× Nonidet P-40 (NP-40) lysis buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 10% glycerol, and 1% NP-40) supplemented with protease and phosphatase inhibitor cocktails (from Sigma-Aldrich and Roche Applied Sciences, respectively), and the lysates were centrifuged at 15,000 rpm at 4°C for 15 min. Supernatants were collected and mixed with the half amount of 3× Laemmli buffer supplemented with 6% 2-mercaptoethanol, followed by incubation at 95°C for 5 min. Aliquots were electrophoresed on an SDS-polyacrylamide gel and transferred to a Protran nitrocellulose membrane (Whatman, Dassel, Germany). The membrane was blocked with Tris-buffered saline containing 0.05% Tween 20 (TBST) supplemented with 5% skim milk, and incubated with primary antibodies diluted in TBST, followed by incubation with HRP-conjugated secondary antibodies. Then, the blots were analyzed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA). If necessary, primary and secondary antibodies were diluted in Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Osaka, Japan). Densitometric analyses were carried out using Image J software.

RNAi experiments
To knockdown endogenous NRDc expression in gastric cancer cells, we used the BLOCK-iT For gene silencing of ADAM17 and ADAM10, we used the Stealth Select RNAi (Invitrogen) according to the manufacturer's instructions. Briefly, non-targeting negative control siRNA (12935-300, from Invitrogen) or siRNA duplex targeting ADAM17 (HSS110434) or ADAM10 (HSS100165) were introduced to TMK-1 cells grown in the growth medium without antibiotics (final siRNA concentration: 10 nM) using Lipofectamine RNAiMAX (Invitrogen).

In vitro cell proliferation analysis
Cell proliferation was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS; Promega, Madison, WI). Briefly, 1.5 or 3.0 × 10 3 cells were seeded in 96-well plates and cultured in the growth medium containing 10% fetal bovine serum for 48-96 h (when indicated, cells were cultured for 24 h after seeding, followed by treatment with reagents such as neutralizing antibodies, small molecule inhibitors, or recombinant proteins for an additional 24 h). Then, 20 µl of MTS tetrazolium compound reagent was added to each well. After the plate was incubated at 37°C for 4 h in 5% CO 2 atmosphere, absorbance at 492 nm was measured using a plate reader. All experiments were performed in octuplicate, and results are shown as mean ± SD of values excluding the minimum and maximum ones.

TACE activity assay
Protease activity of ADAM17, also named as TACE, was analyzed using the InnoZyme TACE Activity Kit (Merck/Calbiochem), except for the substrate. The internally quenched fluorescent TACE substrate used was MCA-PLAQAV-Dpa-RSSSR-NH2 (R&D Systems). Its sequence is derived from pro-TNF-α. After the substrate is cleaved by peptidases at the A-V amide bond, the fluorophore MCA is released from the quenching molecule Dpa. Briefly, cells grown in 10-cm dishes were lysed with ice-cold 1× NP-40 lysis buffer (not supplemented with any protease inhibitor) and centrifuged. One hundred microliters of cell lysates (protein concentration: 3 mg/ml) or recombinant human ADAM17 protein solution (for the positive control) were added to 96-well plates coated with anti-human ADAM17 antibody. After incubation at room temperature for 1 h, the plates were washed 5 times with the provided wash buffer. Then, 100 µl of 10 µM substrate solution was added to each well, and the plates were incubated at 37°C for 5 h. Fluorescence was measured using a fluorescence plate reader (excitation, 355 nm; emission, 460 nm).

Gene expression microarray analysis
Gene expression profiles were compared between the control and NRDc-KD TMK-1 cells using the Affymetrix GeneChip Human Gene 1.0 ST Array (Affymetrix, Santa Clara, CA) with the protocol of the Whole Transcript Sense Target Labeling Assay. Total RNA was extracted from the control or NRDc-KD #1 TMK-1 cells using the RNeasy Mini kit (Qiagen) and digested with DNase. Quality of the extracted RNA was analyzed using the Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). After hybridization with biotinylated sense-strand DNA generated using WT Sense Target Labeling and Control Reagent (Affymetrix), GeneChips were scanned using the GeneChip Scanner 3000 7G System. Microarray data were analyzed using the Affymetrix GeneChip Command Console Software (Affymetrix) and GeneSpring GX Version 10.0.2 (Agilent Technologies). The microarray data from this publication have been submitted to the NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE29114.