A role for aberrantly expressed nuclear localized decorin in migration and invasion of dysplastic and malignant oral epithelial cells

Abstract

Background: Oral cancer is the sixth most common malignancy worldwide with a mortality rate that is higher than many other cancers. Death usually occurs as a result of local invasion and regional lymph node metastases. Decorin is a multifunctional proteoglycan of the extracellular matrix that affects the biology of various types of cancer. Previously; we have shown that decorin is aberrantly expressed in the nucleus in human dysplastic oral keratinocytes (DOK) and malignant squamous cells carcinoma (SCC-25) and human biopsy tissues. In this study, we examined the role of nuclear decorin in oral cancer progression.

Materials and methods: We have used a post-transcriptional gene silencing (RNA interference) approach to stably knockdown nuclear decorin gene expression in DOK and SCC-25 cells using a specific shRNA plasmid and a combination of immunological and molecular techniques to study nuclear decorin function in these oral epithelial cell lines.

Results: More than 80% decorin silencing/knockdown was achieved as confirmed by real time PCR and western blot analysis in both DOK and SCC-25 cells. This RNA interference-mediated knockdown of nuclear decorin expression resulted in significantly reduced invasion and migration in these cell lines as measured by Matrigel™ coated and uncoated Trans well chamber assays respectively. Decorin silencing also resulted in reduced IL-8 mRNA and proteins levels in these cell lines. Culturing decorin silenced DOK and SCC-25 cells, with recombinant human IL-8 or IL-8 containing conditioned medium from respective un-transfected cells for 24 h prior to migration and invasion experiments, resulted in the salvation of reduced migration and invasion phenotype. Furthermore, we found that nuclear localized decorin interacts with EGFR in the nuclear fractions of both DOK and SCC-25 cells. Interestingly, EGFR (trans) activation has previously been shown to be involved in IL-8 production in various epithelia.

Conclusions: Taken together, our results indicate that nuclear localized decorin plays an important role in migration and invasion of oral cancer cells and thus may present as a novel potential target for the treatment of oral cancer.

Figure 1

Validation of stable knockdown of decorin in DOK and SCC-25 cells. DOK and SCC-25 cells were stably transfected with decorin-shRNA (DCN-shRNA), or scrambled sequence-shRNA (Ctrl-shRNA) or no transfection control (WT). A, RNA was extracted and cDNA was subjected to quantitative RT-PCR, normalized decorin expression from one representative experiment of three. B, Nuclear lysates were extracted and were subjected to SDS-PAGE followed by immunoblotting with anti-decorin and anti-β-tubulin antibodies. Data presented is one representative immuoblot of at least three experiments. *** p < 0.001 compared to respective controls.

Figure 2

Decorin silencing does not affect DOK or SCC-25 cell growth/proliferation. WT, control and decorin silenced DOK and SCC-25 cells were cultured for 24 h. During the last hour of culture, 20 μl of CellTiter 96^® Aqueous One Solution Reagent containing a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES) was added to the media (100 μl per well), and color changes were recorded by absorbance at 490 nm. Data are presented as mean ± SE of three replicates of one representative experiment of three.

Figure 3

Migration and invasion suppression in decorin silenced cell lines. A, Cell motility through uncoated filters (migration) was measured 22 h after plating. The migrating cells were fixed, stained, and photographed as described in materials and methods. Each panel represents one representative field of five from duplicate filters of three experiments. B, Migrated cells in each one of the five fields of duplicate filters were counted, numbers represent mean ± SD of three experiments. C, Cells that invaded across the Matrigel™ layer were fixed, stained, and photographed. Each panel represents one representative field of five from duplicate filters of three experiments. D, Migrated and invaded cells in five fields of duplicate filters were counted and % invasion was calculated as described in materials and methods. Numbers represent mean ± SD of three individual experiments. ** p < 0.01, *** p < 0.001 compared to respective controls.

Figure 4

Reduced IL-8 production in decorin Silenced DOK and SCC25. A, RNA was extracted from WT, control and decorin silenced DOK and SCC-25 cells and cDNA was subjected to quantitative RT-PCR, normalized IL-8 expression from one representative experiment of three. B, Cells were cultured and IL-8 was measured in 24 h culture supernatants using ELISA. Data are presented as mean ± SD of three replicates of one representative experiment of four. ** p < 0.01, *** p < 0.001 compared to respective controls.

Figure 5

Migration and invasion suppression rescued by addition of IL8 in decorin silenced cell lines. Decorin silenced DOK and SCC-25 cells were cultured with 2.5 ng/ml of recombinant human IL-8 (R&D Systems, Minneapolis, MN) or conditioned medium (CM) from respective wild type cell lines for 24 h prior to migration and invasion assay. A, Cell motility through uncoated filters (migration) was measured 22 h after plating. The migrating cells were fixed, stained, and photographed as described in materials and methods. A, Migrated cells in each one of the five fields of duplicate filters were counted, numbers represent mean ± SD of three experiments. B, Cells that invaded across the Matrigel™ layer were fixed, stained, and photographed. Migrated and invaded cells in five fields of duplicate filters were counted and % invasion was calculated as described in materials and methods. Numbers represent mean ± SD of three individual experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to respective controls.

Figure 6

Nuclear Decorin associates with EGFR. Nuclear lysates were extracted from DOK and SCC-25 cells and were immunoprecipitated (IP) with decorin antibody. Immunocomplexes were subjected to SDS-PAGE and were analysed by immunoblotting (IB) with anti-decorin and anti-EGFR antibodies.