Background
Oral squamous cell carcinoma (SCC) is the sixth most common cancer in the world [
1,
2]. It accounts for roughly quarter of a million newly diagnosed cancers worldwide, and is the most frequently diagnosed cancer in developing countries of the world [
3,
4]. Despite improvements in surgical techniques, radiation therapy protocols, and chemotherapeutic regimens [
5], the overall five year survival rate for oral SCC remains at 50% and has not significantly improved in the past 30 years. Morbidity associated with the disease further affects the quality of life in patients as nutrition, speech and general immunity gets compromised as well. In oral cancer patients, death usually occurs as a result of local invasion into the stromal tissue of head & neck and cervical lymph node metastases [
6,
7].
Decorin is a member of small leucine-rich repeat proteoglycans (SLRPs) family and is primarily synthesized by fibroblasts and myofibroblasts [
8]. Members of SLRPs family are structurally related and play major roles in the organization of the extracellular matrix (ECM) and the regulation of cell behaviour [
9]. SLRPs have a 40-50 kDa protein core with a central leucine rich repeat (LRR) domains characterized by a common molecular architecture adapted for protein-protein interaction [
10]. Decorin is a known ligand for epidermal growth factor receptor (EGFR) [
11,
12].
Decorin is normally present in the extracellular stromal compartment and has been shown to restrain growth of many tumor cells [
13] by down-regulating the epidermal growth factor receptor (EGFR) [
9]. The underlying mechanism is not fully understood but includes direct decorin binding to EGFR followed by sustained internalization of EGFR via caveolar-mediated endocytosis [
14] and the apoptosis triggering via caspase-3 activation [
15]. Although, decorin is expressed in tumor stroma but it's rarely expressed by cancer cells and tissues themselves as has been demonstrated by analysis of a variety of tumors including colon, pancreas, prostate, lung, ovarian, breast cancer [
16‐
19]. However, studies by us and others have shown that decorin is expressed by oral squamous cell carcinoma and osteosarcoma cells [
20,
21]. Osteosarcoma cells were shown to be insensitive to decorin-induced growth arrest, rather decorin seemed to be beneficial, since it was necessary for osteosarcoma cell migration [
22].
Interleukin (IL)-8, a proinflammatory cytokine of the CXC chemokine family that was originally classified as a neutrophil chemoattractant, has been shown to play a significant role in tumor progression and metastasis in a variety of human cancers, including oral cancers [
23,
24]. This chemokine activates multiple intracellular signalling pathways downstream of two cell-surface, G protein coupled receptors; CXCR1 and CXCR2. IL-8 biologic activity in tumors and the tumor microenvironment contributes to cancer progression through its functions in the regulation of angiogenesis, cancer cell growth and survival, tumor cell invasion and metastasis, leukocyte infiltration and modification of immune responses [
23,
24]. Exogenous decorin has been shown to induce IL-8 production in endothelial cells [
25].
Previously; we have shown that decorin is aberrantly expressed as well as translocated to the nucleus in dysplastic oral keratinocytes (DOK) and malignant squamous cell carcinoma (SCC-25) and in oral cancer biopsy tissue [
20]. In the present study; using shRNA expression plasmid mediated
in vitro stable gene silencing in DOK and SCC-25 cells, we demonstrate that nuclear localized decorin knockdown suppresses migration and invasion in dysplastic and malignant oral epithelial cells. Our data also indicates that reduced migration and invasion phenotype of these cells is mediated by IL-8 modulation as demonstrated by supplementation/rescue experiments. Furthermore, we show here that this nuclear localized decorin interacts with EGFR in the nuclear fractions of both DOK and SCC-25 cells, thus providing the first indication of a unique phenomenon in oral epithelial cells.
Materials and methods
Cell Lines and culture conditions
Oral epithelial origin, premalignant- Dysplastic Oral Keratinocyte (DOK) and malignant- Squamous Carcinoma Cell (SCC-25) lines were routinely maintained in DMEM/F12 (Hyclone, Logan, Utah) supplemented with 10% Foetal Calf Serum for use as
in vitro model in our studies, as described previously [
26,
27]. DOK and SCC-25 cells (5 × 10
5 cells/well) were cultured in complete medium for 24 h, conditioned medium was harvested and was concentrated using a Centriplus centrifugal concentrator (Millipore, Bedford, Massachusetts).
Decorin knock down in DOK and SCC-25 cells in vitro
Silencing of decorin gene expression was achieved using short hairpin RNA (shRNA) technology. Oligonucleotides targeting decorin transcript variants -A1 (RefSeq accession no NM_001920, at nucleotide position 720-740) and -A2 (RefSeq accession no NM_ 133503.2, at nucleotide position 566-586) and scrambled sequence non specific to any gene were custom synthesized, annealed, and cloned into the shRNA expression vector pGeneClip Puro™ (Promega) by Super Array Bioscience Corporation (Frederick, MD). BLAST queries were performed to ensure that the sequences have no significant homology with any other human genes. The transformation grade shRNAi plasmids were amplified in E. coli cultures, purified using Midiprep kits for endotoxin-free DNA vectors and then transfected into DOK and SCC-25 cells using Effectene™ transfection reagent following manufacturer's protocol (Qiagen, Valencia, CA). The stable transfectants were selected for puromycin (Calbiochem, San Diego, CA) antibiotic resistance at 2.5 μg/ml final optimal concentration. To avoid clone-specific variances, pools of stable transfectants (maintained at 1 μg/ml of puromycin) were used in all subsequent experiments. Decorin expression levels were determined at transcript and protein level by quantitative real-time reverse transcription-PCR (RT-PCR) and Western blotting, respectively. Hereafter, untransfected DOK and SCC-25 cells will be referred to as wild type (WT), scrambled shRNA stable transfectants as control (or Ctrl-shRNA in figures), and decorin shRNA stable transfectants as decorin silenced (or DCN-shRNA in figures).
Real-time PCR
RNA was extracted from DOK and SCC-25 cells using RNeasy Plus mini kit (Qiagen, Valencia, CA) and 2.5 μg of total RNA was used to synthesize cDNA, using SuperScript III Reverse Transcriptase (Invitrogen, San Diego, CA). Quantitative RT-PCR was performed using QuantiTect™ SYBR Green PCR kit (Qiagen, Valencia, CA) on the Mini Opticon™ Real-Time PCR system (BioRad, Hercules, CA) as per manufacturer's protocol. Quantitative PCR primer pairs were designed for SYBR Green chemistry based detection of amplicons for DCN (5'-GGACCGTTTCAACAGAGAGG-3', 5'-GACCACTCGAAGATGGCATT-3'), IL-8 (5'-TCTGCAGCTCTGTGTGAAGG-3', 5'-TAATTTCTGTGTTGGCGCAG-3'), and GAPDH (5'-ACAGTCAGCCGCATCTTCTT-3', 5'-GTTAAAAGCAGCCCTGGTGA-3'). GAPDH was used as relative house- keeping gene expression control to normalize for sample variations.
Cell proliferation assay
Cell proliferation was measured using CellTiter 96® Aqueous One Solution -Cell Proliferation MTS based assay (Promega, Madison, WI) according to manufacturer's instructions. Briefly, WT, control and decorin silenced DOK and SCC-25 cells (105 cells/well), were cultured in 96-well flat-bottom plates at a final volume of a 100 μl for 24, 48, and 72 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 each well. Increase in absorbance at 490 nm wavelength (indicating cell proliferation) was measured using a 96-well plate reader (SPECTRAMax 190, Molecular Devices, Sunnyvale, CA) and results were analyzed by SOFTMax Pro software.
Co-Immunoprecipitation and Western Blot Analysis
Cells were rinsed with ice-cold PBS and were lysed in a buffer containing 20 mM Tris, pH 7.6, 0.1% SDS, 1% Triton-X, 1% deoxycholate, 100 μg/ml PMSF, and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Lysates were centrifuged at 20,000 × g for 20 min at 4°C. Nuclear extracts were prepared by using NE-PER kit reagents (Pierce, Rockford, IL) following manufacturer's protocol. Protein concentration was determined by Bis-Cinchonic Acid (BCA) protein assay (Pierce, Rockford, IL) and subjected to 10% SDS-PAGE analysis, followed by transfer to polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The membranes were immunoprobed with 1:500 dilution of monoclonal anti-human decorin antibody (Abcam, Cambridge, MA) or 1:1000 dilution of anti-human β-tubulin polyclonal antibody. Western blots were developed with appropriate horseradish peroxidase conjugated secondary antibodies (Bio-Rad) and ECL Plus chemiluminescence system (Amersham, Arlington Heights, IL) and exposed to auto radiographic films. Radiographs were scanned and densitometry analysis was done using AlphaEase FC software (Alpha Innotech Corporation, San Leandro, CA). For analysis of nuclear lysates by coimmunoprecpitation, Dynabeads® Protein G (Invitrogen, Carlsbad, CA) were used following manufacturer's protocol. Briefly, 5 μg of anti-decorin antibody was coupled to Dynabeads Protein G and then incubated with nuclear lysates to capture the target protein (decorin). Immunocomplexes were eluted and were resolved on 10% SDS-PAGE gel and analyzed by western blotting (as above) using decorin, EGFR antibodies (Abcam).
ELISA for IL-8 Quantification
Decorin silenced, control and WT DOK and SCC-25 cells (5 × 105 cells/well) were cultured in complete medium in 24-well flat-bottom plates at a final volume of a 500 μl. Culture supernatants were collected and IL-8 was assayed with 100 μl of cell free culture supernatant using DuoSet IL-8 ELISA kit (R&D Systems, Minneapolis, MN) according to manufacturer's instructions. Absorbance was read at 450 nm with the SPECTRAMax 190 microplate spectrophotometer and results were analyzed by SOFTMax Pro software (Molecular Devices, Sunnyvale, CA). Sample concentrations were determined by interpolation from the standard curve. IL-8 detection limit was found to be 5.6 pg/ml. Samples were read in triplicate.
Cell Migration and Invasion Assay
The ability of cells to migrate across control inserts (migration) or invade across Matrigel™- coated inserts (invasion) was assayed using BD Falcon control inserts or BD BioCoat Matrigel™ invasion chambers (BD Biosciences, San Jose, CA), respectively. The BD BioCoat Matrigel™ invasion chambers consist of BD Falcon tissue culture companion plate with Falcon cell culture inserts containing 8 micron pore size PET membrane, pre-coated with a thin layer of Matrigel™ basement membrane matrix. Manufacturer's instructions were followed to perform the assay. Briefly, serum free DMEM/F12 medium (0.5 ml) containing 105cells were added to the upper chamber, and 0.75 ml of DMEM/F12 medium containing 10% serum was added to the lower chamber as a chemo-attractant. After overnight incubation at 37°C and 5% CO2, cells on the upper surface of the filter (cells that had not penetrated the filter) were removed using a cotton swab. Cells that had migrated to the lower surface of the filter were fixed in 100% methanol and stained with 0.005% crystal violet. For each filter, the number of migrated cells in 5 medium-power fields (magnification of 20×) was counted using bright field microscopy, and photographed. Assays were performed in duplicates and repeated at least three times. Invasion index is expressed as percentage of invading cells, and is calculated by dividing mean number of cells invading through Matrigel™ membrane over mean number of cells migrating through the non-coated control insert membrane per microscopic filed over five fields per assay, and ratio then multiplied by 100 for percent values.
Statistical Analysis
Student's paired t test was used to determine the statistical significance of the data. Statistical analysis was performed on Graph Pad Prism Software. Significance was evaluated at p values: * p < 0.05, ** p < 0.01, *** p < 0.001.
Discussion
Decorin, a multifunctional molecule of the extracellular matrix, has been assigned a plethora of functions. Among these, the most intriguing is the ability to inhibit growth and metastasis of a wide range of cancer cells in vitro. Functions of decorin as a naturally occurring oncosuppressive agent are being studied in detail. Decorin is normally present in the extracellular stromal compartment and analysis of a variety of tumours indicates that it is seldom expressed by cancer tissue. However studies by us and others have demonstrated that there are exceptions to this prevailing decorin expression and decorin mediated growth suppression model in cancer. In our previous studies of oral precancerous and cancerous lesions and cellular models of oral cancer progression, we demonstrated that decorin is aberrantly expressed and localized in the nucleus in the dysplastic and malignant oral epithelial cells [
20]. In this study we used shRNA expression plasmid mediated
in vitro stable gene silencing in DOK and SCC-25 cells to study the role of nuclear localized decorin in oral cancer.
The ability of a cancer cell to undergo migration and invasion allows it to change position within the tissues allowing neoplastic cells to enter lymphatic and blood vessels for dissemination into the circulation, and then undergo metastatic growth in distant organs. Using an
in vitro Matrigel™ coated and uncoated Trans well chamber based invasion and migration assay, we find here that nuclear decorin silencing results in impaired invasive and migratory potential in both dysplastic and malignant oral epithelia. It is interesting to note that in osteosarcoma cells, decorin was also found to be necessary for cell migration [
21]. Moreover, these Osteosarcoma cells have also been shown to constitutively produce decorin and manifest resistance to decorin induced growth arrest.
Growth and motility of various cancer cells is influenced by assorted growth factors, cytokines and chemokines. Recently, there has been increasing evidence that chemokines have a role in tumor biology. Chemokines were first described as small peptides controlling cell migration, especially that of leukocytes during inflammation and immune response. Since then, a broad spectrum of biological activities has been described as chemokine-regulated tumorigenesis that effect tumors and their microenvironment. Increased expression of IL-8 and/or its receptors has been characterized in cancer cells, endothelial cells, infiltrating neutrophils, and tumor-associated macrophages, suggesting that IL-8 may function as a significant regulatory factor within the tumor microenvironment. Many tumor cells, such as prostate carcinoma, melanoma, breast carcinoma and gastric carcinoma cell lines, respond chemotactically to IL-8 [
30‐
34]. IL-8 can also act as an autocrine growth factor for melanoma, colon carcinoma, lung adenocarcinoma and prostate carcinoma cell lines [
35‐
38]. In addition, IL-8 has been shown to be produced by oral squamous cell carcinoma both constitutively and in response to cytokines[
23]. In these oral carcinoma cell lines, IL-8 was shown to have positive influence on migration and invasion via up-regulation of MMP-7. Given the importance of IL-8 in tumor migration and invasion, we tested whether IL-8 was involved in nuclear decorin silencing mediated migration and invasion suppression in our system. We found that, IL-8 production was significantly attenuated in nuclear decorin silenced DOK and SCC25 cells. Rescue of migration and invasion suppression upon addition of exogenous IL8 indicates that reduced IL-8 might be forming the basis of migration and invasion mitigation in these decorin silenced cell lines. Whether or not there is a direct or indirect relationship between decorin and IL-8 is not known currently and needs further exploration. However, intriguingly, decorin has been shown to induce IL-8 production at least in endothelial cells [
25].
We have previously shown that decorin is aberrantly expressed in the nucleus in DOK and SCC-25 oral cell lines and human biopsy tissues [
20]. However, underlying mechanism by which decorin localizes to the nucleus in these cells is unclear. Decorin lacks nuclear localization signal (NLS), hence it might be binding with another protein that carries NLS thus providing a means for decorin to localize to the nucleus in the oral cells. Elucidation of potential interactions of the aberrant decorin expressed in the oral cancer progression model and its biological implications need to be addressed further. As such, in this study we explored the possible nuclear decorin binding partners by immuoprecipitation experiments on nuclear lysates obtained from DOK and SCC-25 cells. Decorin is a known alternate biological ligand for EGFR, other than EGF [
11]. It binds to a discrete region of the EGFR which is partially overlapping but distinct from the EGF-binding domain [
12]. Emerging evidence suggests that EGFR family carries NLS and either full length or fragmented EGFR family members can be shuttled from the plasma membranes to the nucleus [
39‐
41]. This direct mode of the EGFR signaling pathway is distinct from the traditional transduction pathway and involves cellular transport of EGFR from the cell-surface to the cell nucleus, association of nuclear EGFR with transcriptional co-factors and target gene promoters, and transcriptional regulation of the target genes. Aberrant expression and nuclear localization of EGFR has been demonstrated by many different groups in a variety of human cancers including oral cancer [
42‐
44]. Mutant EGFR, a truncated EGFR variant (EGFRvIII), has also been described in head and neck cancer [
45]. In search of a possible mechanism by which decorin is aberrantly localized in the nucleus, we sought out to determine if nuclear decorin interacts with nuclear EGFR in our oral cancer model. We found that EGFR is expressed in the nuclear lysates obtained from DOK and SCC-25 cells, and very interestingly, results of our co- immunoprecipitation experiments demonstrate that nuclear decorin does interacts with nuclear EGFR in these dysplastic and malignant oral epithelia. This indicates a possible means by which decorin is aberrantly localized in the nuclei in our oral cancer model. As mentioned above, nuclear EGFR has been shown to act as transcription factor and its transcriptional activity appears to depend on its C-terminal transactivation domain and its physical and functional interaction with other transcription factors that contain DNA-binding activity [
46]. Interestingly, EGFR (trans) activation has been shown to be involved in IL-8 production in lung and skin epithelia [
28,
47]. Whether or not nuclear EGFR has direct or indirect role in IL-8 transcription is not known currently. Besides decorin-EGFR relation, additional interactions with other nuclear and/or cytosolic factors may result from the aberrant localization of decorin and perhaps result in some of decorin's tumor-promoting role in oral cancer. Together, results from our study suggest the importance of decorin as a potential therapeutic target in oral cancer, as it modulates migration and invasion of premalignant and malignant oral epithelial cells. Further mechanistic studies are warranted to decipher how IL-8 expression is regulated by nuclear localization of decorin and/or its interaction with EGFR in these cells. Studies in our laboratory are underway in this direction which will shed light on additional biological aspects of nuclear localized decorin in oral cancer progression.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
ND contributed to study concept and design and devised, performed, and analyzed the experiments, interpreted data and wrote the manuscript. AGB conceptualized the study design, analyzed results and helped in data interpretation, approved the final manuscript and directed the research program. All authors reviewed and approved the final manuscript.