MDA-9/Syntenin-Slug transcriptional complex promote epithelial-mesenchymal transition and invasion/metastasis in lung adenocarcinoma.

Melanoma differentiation-associated gene-9 (MDA-9)/Syntenin is a novel therapeutic target because it plays critical roles in cancer progression and exosome biogenesis. Here we show that Slug, a key epithelial-mesenchymal-transition (EMT) regulator, is a MDA-9/Syntenin downstream target. Mitogen EGF stimulation increases Slug expression and MDA-9/Syntenin nuclear translocation. MDA-9/Syntenin uses its PDZ1 domain to bind with Slug, and this interaction further leads to HDAC1 recruitment, up-regulation of Slug transcriptional repressor activity, enhanced Slug-mediated EMT, and promotion of cancer invasion and metastasis. The PDZ domains and nuclear localization of MDA-9/Syntenin are both required for promoting Slug-mediated cancer invasion. Clinically, patients with high MDA-9/Syntenin and high Slug expressions were associated with poor overall survival compared to those with low expression in lung adenocarcinomas. Our findings provide evidence that MDA-9/Syntenin acts as a pivotal adaptor of Slug and it transcriptionally enhances Slug-mediated EMT to promote cancer invasion and metastasis.


Yeast two-hybrid
Yeast two-hybrid screening was performed as previously described [1]. Yeast strain L40 cells transformed with pBTM116-Slug were mated with yeast cells, which were transformed with prey genes from human prostate cancer cDNA library in the pACT2 vector, the kindly gift from Dr. Hsiu-Ming Shih (Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan). The resulting mated yeast cells were selected on a medium lacking histidine, leucine, and tryptophan in 384-well plates. Yeast strain AH109 cells were co-transformed with pACT2-Syntenin and pAS2-1-Slug or pAS2-α-catulin and the transformed yeast cells were grown on a yeast dropout media lacking the indicated selection medium. The samples were incubated at 30°C for 5-7 days. Lentiviral vector expressing siRNA against human MDA-9/Syntenin (shSyntenin-a and shSyntenin-b), Slug (shSlug) and its control (shLacZ) were obtained from the RNAi consortium at Academia Sinica. The target sequence of each shRNA to human gene was listed in Supplementary  Table 2. For lentiviral production, HEK293T cells were co-transfected with the indicated lentiviral vector [Flagtagged, untagged Syntenin (WT, ∆PDZ1, ∆PDZ2, PDZs, and PDZS-NLS), Slug, shSyntenin, shSlug and shLacZ] with two helper plasmids (pMD.G and pCMV∆8.91) using Lipofectamine 2000 reagents according to the manufacturer's protocols.

Transfection and viral infection
Virus-containing medium was collected at 24, 48, and 72 h post-transfection, centrifuged, and filtered through 0.45 mm-pore-size filters. HEK293, CL1-5 and CL141 cells were infected with the indicated lentivirus in media containing polybrene (8 μg/ml). At 24 h postinfection, the cells were replaced with fresh medium for 48 h and then used for all of the experiments.
The beads were then collected and washed three times in IP lysis buffer and eluted by boiling in SDS sample buffer. The protein elutes were separated by SDS-PAGE and processed for immunoblotting using the relevant primary antibodies.

Modified boyden chamber invasion assay
Modified Boyden chambers with polycarbonatemembrane inserts (pore size 8 mm; BD Falcon, Franklin Lakes, NJ) and coated with Matrigel (BD Biosciences) (12 μg for HEK293 and 30 μg for CL1-5 and CL141) were performed for cell invasion assays. Cells (2.5 × 10 4 ) suspended in DMEM or RPMI medium containing 10% Nu-Serum (Invitrogen) were plated in the upper chambers. After 24 h incubation at 37°C, the cells were fixed with methanol and stained with a 50 μg/ml solution of propidium iodide (Sigma-Aldrich, MO, USA) at room temperature for 30 min. Each membrane was photographed and the number of cells counted under a microscope at 650 magnification using the Analytical Imaging Station software package (Imaging Research Inc, St Catherine's, Canada). Each experiment was assayed in triplicate.

Cell fractionation
The MDA-9/Syntenin and Slug distribution in cells were analyzed by Qproteome Cell Compartment Kit (Qiagen) according to the manufacturer's protocol. The cells were scraped and washed by cold PBS twice. The cytosolic, membrane, and nuclear fractions were obtained from centrifuged cell pellets incubated with the extraction buffers at 4°C. The proteins of the cell fraction were detected by SDS-PAGE and by immunoblotting.

Reverse-transcriptase PCR
The total RNA of cells was extracted by Trizol (Invitrogen) and cDNAs were prepared and underwent PCR as described previously. The primer sequences used were listed in Supplementary Table 3. The PCR product of each sample was determined by agarose gel electrophoresis.

Expression of GST fusion proteins and GST pull-down assay
The recombinant GST-Syntenin and its deletion derivatives were induced in XA-90 bacterial strain with 0.5 mM IPTG for 30 minutes, and then purified with glutathione beads (GE Healthcare/Amersham Biosciences, Buckinghamshire, UK) according to standard protocol. HA-Slug and HDAC1-Flag were produced by TNT ® Quick Coupled Transcription/Translation Systems (Promega, Madison, WI). HA-Slug or HDAC1-Flag was mixed with purified GST-Syntenin or its variants. Pull-down assays were performed at 4°C for 1 h. The beads were then washed thoroughly with PBS buffer. The bound proteins were eluted by boiling in SDS sample buffer, separated by SDS-PAGE, and detected by immunoblotting.

Immunofluorescence
The transfected cells that expressed EGFP-Syntenin and DsRed-Slug were fixed with methanol/acetone (1:1) for 5 min and washed with PBS. All of the samples were mounted onto microscope slides with ProLong Gold antifade reagent with DAPI (Invitrogen Life Technologies) and examined and photographed by an LSM 510 laser scanning confocal microscope (Carl Zeiss).