Abstract
Squamous cell carcinoma is a prevalent head and neck tumor with high mortality. We studied the role played by laminin α1 chain peptide AG73 on migration, invasion, and protease activity of cells (OSCC) from human oral squamous cell carcinoma. Immunohistochemistry and immunofluorescence analyzed expression of laminin α1 chain and MMP9 in oral squamous cells carcinoma in vivo and in vitro. Migratory activity of AG73-treated OSCC cells was investigated by monolayer wound assays and in chemotaxis chambers. AG73-induced invasion was assessed in Boyden chambers. Invasion depends on MMPs. Conditioned media from cells grown on AG73 was subjected to zymography. We searched for AG73 receptors related to these activities in OSCC cells. Immunofluorescence analyzed AG73-induced colocalization of syndecan-1 and β1 integrin. Cells had these receptors silenced by siRNA, followed by treatment with AG73 and analysis of migration, invasion, and protease activity. Oral squamous cell carcinoma expresses laminin α1 chain and MMP9. OSCC cells treated with AG73 showed increased migration, invasion, and protease activity. AG73 induced colocalization of syndecan-1 and β1 integrin. Knockdown of these receptors decreased AG73-dependent migration, invasion, and protease activity. Syndecan-1 and β1 integrin signaling downstream of AG73 regulate migration, invasion, and MMP production by OSCC cells.
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Introduction
Squamous cell carcinoma represents 95% of oral neoplasms and constitutes an important health problem specially in developing countries, as survival index tends to be small (50%) compared to others malignant tumors [1, 2]. Cell alterations related to oral squamous carcinoma have consumption of tobacco and alcohol as major promoting factors [3].
Microenvironment of carcinoma is formed not only by neoplastic cells, but also by surrounding stroma [4–6]. Therefore, its progression requires tumor cell interactions with extracellular matrix (ECM), a three-dimensional network that functions as structural scaffold and instruction source for cells [7]. In the epithelium, cells form a specialized ECM sheet-like structure, the basement membrane, composed by laminin, type IV collagen, nidogen, and perlecan [8].
We previously demonstrated the influence of laminin, an important basement membrane protein, regulating salivary gland tumors [9–16]. These results prompted us to address the role played by this molecule on squamous cell carcinoma.
Laminins are heterotrimeric glycoproteins prominently expressed in basement membranes that promote cell adhesion, migration, growth, and differentiation [17, 18]. Like other ECM molecules, laminin undergoes controlled MMP processing, generating small fragments and bioactive peptides that can influence cell behavior [19, 20].
We have been studying effects of different bioactive peptides derived from laminin-111, former laminin-1 [17], in tumor biology [9, 11–14, 16]. We are particularly interested in the peptide AG73 (RKRLQVQLSIRT) from the LG4 domain of laminin α1 chain [21–23]. AG73 is relevant in tumor biology, being related to liver and ovarian cancer metastasis, lung colonization of melanoma cells, and angiogenesis promotion [24–26]. These evidences suggest that AG73 may have different roles in promoting tumor growth and metastasis. Previously, we have shown that this peptide regulates morphology, adhesion, and protease activity of an adenoid cystic carcinoma cell line [14]. This regulation would depend on syndecan-1 and β1 integrin [14]. However, the effects of AG73 in oral squamous cell carcinoma have never been assessed.
In this paper, we studied the role played by AG73 regulating migration, invasion, and protease activity of cells (OSCC) from human oral squamous cell carcinoma. We also addressed whether syndecan-1 and β1 integrin would regulate AG73-induced effects.
Materials and methods
Immunohistochemistry
Five cases of oral squamous cell carcinoma were studied by immunohistochemistry. Table 1 shows distribution of cases according to demographic, lifestyle, and clinico-pathological variables. Sections (3 µm) obtained from formalin-fixed paraffin-embedded tissues were subjected to Envision System (Dako, Carpinteria, CA, USA) and mounted on 3-aminopropyltriethoxysilane-coated slides (Sigma Chemical Corp, St Louis, MO, USA). Sections were dewaxed in xylene, hydrated in graded ethanol, and incubated in 3% H2O2 in methanol for 20 min to inhibit endogenous peroxidase activity. Antigen retrieval was carried out with citrate buffer (pH 6.0) in a Pascal chamber (Dako) for 30 s. Sections were blocked for 1 h with 1% bovine serum albumin (BSA, Sigma) in phosphate-buffered saline (PBS). To detect laminin α1 chain, rabbit antiserum HK-175 (kindly provided by Dr. Hynda Kleinman, NIDCR, NIH, USA) was used. MMP9 was labeled by a mouse monoclonal antibody (IM37L, Calbiochem Inc., San Diego, CA, USA). All primary antibodies were diluted 1:50 in PBS and incubated for 1 h. Diaminobenzidine (Sigma) was used as chromogen and sections were counterstained with Mayer's hematoxylin (Sigma). Replacement of primary antibodies by non-immune sera served as negative controls.
We also analyzed presence of laminin α1 chain and MMP9 in similar areas of oral squamous cell carcinoma. Serial sections were subjected to the same immunohistochemistry protocol described above.
Cell culture
OSCC cell line, derived from human oral squamous cell carcinoma, was kindly provided by Dr. Ricardo B. Azevedo (Morphology and Genetics Department, UnB, Brazil). OSCC characterization was published elsewhere [27]. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) supplemented by 10% fetal bovine serum (Cultilab, Campinas, SP, Brazil) and 1% antibiotic–antimycotic solution (Sigma) and kept in a humidified atmosphere of 5% CO2 at 37°C.
Peptides
EZ Biolab (Westfield, IN, USA) synthesized AG73 (RKRLQVQLSIRT) and the scrambled control peptide AG73SX (RTLRIKQSVRLQ). Peptides purity was 98% (RP-HPLC), with molecular weight confirmed by mass spectrometry. Throughout the manuscript, treated samples will be designated AG73 while controls will be named AG73SX.
Immunofluorescence
OSCC cells were grown on glass coverslips for 24 h. To detect laminin α1 chain, culture medium was removed and cells were incubated with rabbit antiserum HK-175 diluted 1:50 in 5% non-fat milk, 0.1 M sodium chloride, and 0.02 M sodium phosphate (pH 7.4) for 1 h on ice. After rinsing with 0.15 M NaCl, 0.05 M sodium phosphate (pH 7.4), cells were fixed in 3% paraformaldehyde for 20 min at room temperature. Alexa-568 anti-rabbit secondary antibody (Invitrogen-Molecular Probes, Carlsbad, CA, USA) revealed primary antibody. Replacement of primary antibody by non-immune serum served as negative control.
To stain for MMP9, cells were fixed and permeabilized with 0.05% Triton X-100 (Sigma) in PBS for 5 min, and blocked with 10% goat serum. Samples were incubated with anti-MMP9 mouse monoclonal antibody (IM37L, Calbiochem) diluted 1:250 in PBS with 2% goat serum. Anti-mouse Alexa-568 secondary antibody (Invitrogen-Molecular Probes, Carlsbad, CA, USA) revealed primary antibody. Replacement of primary antibody by non-immune serum served as negative control.
We investigated colocalization of laminin α1 chain and MMP9 in OSCC cells. Cells were fixed and double-stained with the same antibodies described above. No permeabilization step was carried out. Appropriated fluorescent secondary antibodies revealed primary antibodies.
Colocalization analysis of syndecan-1 and β1 integrin in OSCC cells treated by AG73 was carried out. Cells in serum-free media were cultured on glass coverslips coated with either AG73 or AG73SX (100 µg/ml). After 6 h in culture, cells were double-labeled to syndecan-1 and β1 integrin. Initially, culture medium was removed and cells were incubated with anti-syndecan-1 mouse monoclonal antibody (CBL 588, Chemicon Inc, Temecula, CA, USA) diluted 1:100 in 5% non-fat milk, 0.1 M sodium chloride, 0.02 M sodium phosphate (pH 7.4), for 1 h on ice. Cells were then rinsed with 0.15 M NaCl, 0.05 M sodium phosphate (pH 7.4) and fixed in 3% paraformaldehyde for 20 min at room temperature. Alexa-568 anti-mouse secondary antibody (Invitrogen-Molecular Probes) revealed syndecan-1 primary antibody. For β1 integrin staining, cells were permeabilized with 0.05% Triton X-100 (Sigma) for 2 min before incubation with β1 integrin rabbit polyclonal antibody (AB1952, Chemicon) diluted 1:100 in 5% non-fat milk, 0.1 M sodium chloride, 0.02 M. sodium phosphate. Anti-rabbit Alexa-488 secondary antibody revealed β1 integrin primary antibody.
In colocalization analysis, a minimum of 50 cells was studied and results repeated consistently.
Monolayer wound assay
Migration of OSCC cells was investigated through an in vitro monolayer wound assay [16, 28]. Cells grown in 24-well plates in DMEM to confluence were scraped with pipette tip to create a cell-free area. Wounded monolayers were washed with serum-free media to remove cell debris and incubated with serum-free DMEM containing either AG73 or AG73SX (100 µg/ml). Wound closure was followed after 0, 24, and 48 h. Reference points were added to the bottom of each well, allowing photograph acquisition of identical fields each time. Wound healing effect was calculated as percentage of remaining cell-free area compared to initial wound area (arbitrarily set as 100%). Experiments were carried out in triplicate at least three times.
All monolayer wound assays included non-peptide controls. Positive control was DMEM with 10% fetal bovine serum (FBS). Negative control was serum-free DMEM.
Migration assay
To confirm results of monolayer wound assays, migration assays were also carried out in ten-well chemotaxis chambers (Neuro Probe Inc., Gaithersburg, MD, USA), using porous polycarbonate membrane (8 µm pore size). OSCC cells (105) were plated into the upper chamber in serum-free DMEM. The lower chamber was filled with serum-free DMEM containing either AG73 or AG73SX (100 µg/ml). Cells were cultured in these conditions for 20 h. Polycarbonate membrane was fixed with 4% paraformaldehyde, and cells on the upper side of membrane were removed with cotton swabs. Migrated cells, located on the lower side, were stained with crystal violet, photographed, and counted at a final magnification of 500×. On each well, seven random fields were evaluated. Each experiment was carried out in triplicates at least three times.
OSCC cells had syndecan-1 and β1 integrin silenced by small interfering RNA (siRNA; Santa Cruz Biotechnology, San Ramon, CA, USA). Cells transfected with scrambled siRNA (Santa Cruz) served as controls. As specificity control, OSCC cells were transfected with β3 integrin siRNA (Santa Cruz). Cells with reduced expression of receptors were treated either with AG73 or AG73SX and subjected to migration assay as described above. Experiments were carried out in triplicates at least three times.
All migration assays included non-peptide controls. Positive control was DMEM with 10% fetal bovine serum (FBS). Negative control was serum-free DMEM.
Invasion assay
Invasion assays were carried out in ten-well Boyden chambers (Neuro Probe), using polycarbonate membrane (8 µm pore size) coated with Matrigel (13 mg/ml). OSCC cells (15 × 104) were plated into the upper chamber in serum-free DMEM. The lower chamber was filled with medium containing either AG73 or AG73SX in different concentrations (25, 100, 250, and 500 µg/ml). Cells were cultured in these conditions for 40 hours, and fixed with 4% paraformaldehyde. Cells on upper side of membrane were removed and invaded cells fixed, stained, counted, and analyzed as described before. Each experiment was carried out in triplicates at least three times.
OSCC cells had syndecan-1 and β1 integrin silenced by siRNA. Cells transfected with scrambled siRNA served as controls. As specificity control, OSCC cells were transfected with β3 integrin siRNA (Santa Cruz). Cells with reduced expression of receptors were treated with either AG73 or AG73SX and subjected to invasion assays. Experiments were carried out in triplicate at least three times.
All invasion assays included non-peptide controls. Positive control was DMEM with 10% fetal bovine serum (FBS). Negative control was serum-free DMEM.
Protease activity of AG73-treated cells
OSCC cells were cultured in six multiwell plates coated with different concentrations (25, 100, 250 and 500 µg/ml) of either AG73 or AG73SX diluted in Milli-Q water and allowed to evaporate overnight in a hood. Cells (104) were plated for at least 24 h to adhere and spread, and washed with PBS. Culture medium was replaced by serum-free media for 24 h. The presence of MMPs in conditioned media was assessed by zymography, as described elsewhere [14]. Experiments were carried out at least three times with consistent results.
OSCC cells had syndecan-1 and β1 integrin silenced by siRNA. Cells transfected with scrambled siRNA served as controls. As specificity control, OSCC cells were transfected with β3 integrin siRNA (Santa Cruz). Conditioned media of cells with reduced expression of receptors and treated with either AG73 or AG73SX were subjected to zymography. Experiments were carried out in triplicate at least three times.
Small interfering RNA
OSCC cells were transfected with commercially available siRNA targeting syndecan-1 and β1 integrin (Santa Cruz) following manufacturer's instructions. One day before transfection, subconfluent OSCC cells were cultured in DMEM with 10% fetal bovine serum and without antibiotic–antimycotic solution. Cells were incubated with a complex formed by siRNA (50 nM), transfection reagent (Lipofectamine 2000, Invitrogen), and transfection medium (Opti-MEM I, Invitrogen) for 30 h at 37°C. A siRNA scrambled sequence (Santa Cruz proprietary target sequence) and a β3 integrin siRNA (Santa Cruz) were used as controls. Transfection efficiency was confirmed by Western blot, as described elsewhere [14].
Statistical analysis
Student’s t test was carried out to evaluate differences between two groups. Differences between three or more groups were assessed by ANOVA, followed Bonferroni’s multiple comparisons test. The software used was GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA).
Results
Oral squamous cell carcinoma and OSCC cells express laminin α1 chain and MMP9
AG73 peptide is present in laminin α1 chain and may depend on MMP processing to be released to cells [19, 20]. Therefore, we searched for laminin α1 chain and MMP9 in human oral squamous cell carcinoma in vivo.
Laminin α1 chain (antibody HK-175) was detected in formalin-fixed paraffin-embedded samples of human oral squamous cell carcinoma. A prominent expression of this protein was found at invasion sites (Fig. 1a). MMP9 was present in cells from squamous cell carcinoma and in stroma (Fig. 1 b and c). Negative controls showed no staining (Fig. 1d).
OSCC cells expressed laminin α1 chain (Fig. 2a) and MMP9 (Fig. 2b). Laminin α1 (antibody HK-175) was found as dots throughout cell membrane (Fig. 2a). Laminin staining represents protein distribution on cell membrane, since samples were not permeabilized. OSCC cells exhibited MMP9 as punctate intracellular staining (Fig. 2b).
Laminin α1chain and MMP9 colocalize in vivo and in vitro
Serial sections showed laminin α1 chain and MMP9 sharing similar distribution in vivo and in vitro. Both proteins were found at invasion sites of human oral squamous cell carcinoma (Fig. 3a). Furthermore, colocalization spots were found in OSCC cells (Fig. 3b).
AG73 increases migration of OSCC cells
Monolayer wound assays (Fig. 4a) showed that OSCC cells treated with AG73 increased migratory activity compared to cells grown on AG73SX. After 24 h, OSCC cells treated with AG73 showed cell-free area decreased to 25%, while cells treated by AG73SX exhibited 75% of cell-free area. After 48 h, only 8% of wound area remained cell-free in AG73 group, compared to a cell-free area of 70% in control scrambled peptide. Monolayer wound healing induced by AG73 was increased compared to non-peptide controls. Migration assays carried out in chemotaxis chambers for 20 h (Fig. 4b) confirmed results obtained with monolayer wound assays. AG73 induced a threefold increase in cell migration compared to controls. Peptide-induced migration was increased compared to non-peptide controls.
Migration experiments were carried out in triplicate at least three times. The results repeated consistently.
AG73 increases OSCC cells invasion and MMP secretion
Invasion assays in Boyden chambers coated with Matrigel showed that AG73 increased invasion of OSCC cells in a dose-dependent manner (Fig. 5a). Peptide-induced invasion was increased compared to non-peptide controls in all but 25 µg/ml concentration.
Invasion of tumor cells to surrounding connective tissue involves activity of matrix metalloproteinases (MMPs) [29]. To address whether invasion was related to MMPs, conditioned media of OSCC cells cultured on either AG73 or AG73SX was subjected to zymography. OSCC cells showed gelatinolytic bands corresponding to molecular weights of MMPs 2 and 9 (Fig. 5b). MMPs 2 and 9 positive controls were analyzed on the same gel to confirm the result. AG73 induced a dose-dependent increase of active MMP9 compared to AG73SX (Fig. 5b). To determine whether these bands were MMPs, zymograms of conditioned media from AG73-treated cells were incubated in the presence of calcium chelator EDTA and heavy metal chelator 1,10-phenanthroline. Both treatments resulted in loss of gelatinase activity demonstrating that gelatinolytic bands were MMPs (not illustrated).
Experiments showed increased protease activity using AG73 either as soluble ligand or as immobilized factor. Invasion and protease activity experiments were carried out in triplicate at least three times. The results repeated consistently.
Syndecan-1 and β1 integrin mediate AG73-induced migration and invasion in OSCC cells
To understand mechanisms involved in AG73-induced migration, invasion, and protease activity, we decided to study putative receptors for this peptide in OSCC cells. Syndecan-1 has already been identified as an AG73 ligand [21]. Furthermore, we have also characterized syndecan-1 and β1 integrin as key players regulating migration, invasion, and protease activity of cells from salivary gland neoplasms [14].
Immunofluorescence showed that OSCC cells express syndecan-1 and β1 integrin (Fig. 6). Furthermore, AG73 induced a polarized distribution of colocalized syndecan-1 and β1 integrin in OSCC cells (Fig. 6a–f). Cells treated by AG73SX control peptide showed no colocalization of these receptors (Fig. 6g–l).
To analyze whether syndecan-1 and β1 integrin would be related to AG73-induced activities, OSCC cells had either syndecan-1 or β1 integrin silenced by siRNA. Cells with reduced receptor expression were treated by either AG73 or AG73SX and subjected to migration and invasion assays and zymography. Silencing of receptors caused significant decrease in AG73-induced migration (Fig. 7a), invasion (Fig. 7b), and protease activity (Fig. 7c) of OSCC cells compared to controls. Effect of these receptors is specific, since knockdown of β3 integrin did not affect AG73-mediated activities in OSCC cells (Fig. 7a–c). Transfection efficiency was confirmed by immunoblot (Fig. 7d).
Discussion
We demonstrated that AG73 induces migration, invasion, and protease activity in cell line derived from oral squamous carcinoma (OSCC). Syndecan-1 and β1 integrin signaling downstream of AG73 probably regulate these events. This peptide has been related to similar biological effects in an adenoid cystic carcinoma cell line [17]. However, to our knowledge, this is the first report establishing the role of a laminin-derived peptide in oral squamous cell carcinoma. Importance of AG73 in different tumors emphasizes its relevance in tumor biology.
It has been established that the surrounding stroma of a neoplasm is related with its biological behavior [7]. During invasion, neoplastic cells need to cross basement membrane and interstitial stroma, becoming exposed to ECM components which may regulate cell function [30].
Laminins are major basement membrane proteins composed of three polypeptide chains (α, β, γ). About 16 laminin isoforms have been identified [17]. Laminin-111 is composed by α1, β1, and γ1 chains and the LG4 globular domain of α1 chain contains AG73 peptide. Laminin α1 chain is expressed in vivo and in vitro in human oral squamous cell carcinoma. Based on these findings, we attempted to establish an in vitro system, reproducing effects of peptide AG73 in different events of oral squamous carcinoma.
We observed expression of laminin α1 chain and MMP9 at invasion sites of human oral squamous cell carcinoma. Furthermore, immunofluorescence showed laminin α1 chain and MMP9 colocalized in OSCC cells. These results may suggest MMP9-mediated breakdown of laminin. Laminins are substrates for MMPs, and controlled proteolytic processing of this molecule generates fragments containing bioactive peptides and cryptic sites [19, 20]. Many bioactive peptides have been shown to regulate cell behavior and cryptic domains contained in the ECM are exposed by proteolysis and stimulate biological responses [19, 20].
Several active sequences on laminin-111 have been identified [22, 23]. The YIGSR sequence located on β1 chain promotes cell adhesion and migration [16, 31]. PDGSR and F-9 (RYVVLPR), from β1 chain, also promote cell adhesion [32]. C16, located on γ1 chain, induces angiogenesis, migration, protease activity, and metastasis in different systems [33]. SIKVAV and AG73, from α1 chain, participate in cell adhesion, proliferation, neurite outgrowth, acinar formation, and protease activity [11–14, 19, 21].
Our results showed that AG73 promotes biological functions on cells derived from oral squamous cell carcinoma. This peptide induced migration of OSCC cells. Previously published data showed that AG73 promotes cell adhesion, spreading, and migration [14, 34]. Active migration of neoplastic cells is a requirement for tumor invasion, and can be influenced by chemoattractants and construction of adhesion pathways [35].
Another relevant step in tumor progression is ECM degradation. Key enzymes in this process are MMPs, a family of over 24 endopeptidases frequently found at high levels in tumors [29, 36]. Our data showed that AG73 enhanced invasion of OSCC cells in a dose-dependent manner. Since invasion involves MMPs-mediated matrix degradation, we explored the role of AG73 regulating MMPs. The peptide induced a dose-dependent increase of active MMP9 compared to controls. Taken together these results suggest that AG73 regulates invasion of OSCC cells through MMP9.
To assess mechanisms underlying AG73 effects in OSCC cells, we analyzed putative receptors for this peptide. Syndecan-1 and β1 integrin are involved in AG73 effects in other systems [14, 21]. Integrins are major laminin receptors that mediate cell adhesion, migration, and differentiation [37]. Overexpression of α2β1 and α3β1 integrins in squamous cell carcinoma has been already described [38, 39]. In vitro studies showed that squamous carcinoma cells use α6β1 integrin to adhere and migrate over laminin-111 [40]. Furthermore, these cells express higher levels of β1 integrin subunit compared to normal keratinocytes [40]. On the other hand, syndecans are a family of heparan sulfate proteoglycans, which synergically cooperate with integrins in the formation of adhesion complexes, cell spreading, and unidirectional migration [41].
OSCC cells with expression of syndecan-1 and β1 integrin silenced by siRNA decreased AG73-related migration, invasion, and protease activity. This result suggests that these receptors may cooperate with AG73 influencing behavior of OSCC cells. Our hypothesis is supported by reports showing direct interaction between syndecans and integrins [42].
The knowledge on biological effects of laminin-derived peptides such as AG73 is fragmented, without a clear sequence and flow of information. We have tried to link biological events, to better understand the role played by this peptide on squamous cell carcinoma. Laminin-derived peptides have important effects on cellular interactions that control adhesion, motility, and invasion of cell lines [43]. Based on our experimental findings, we propose that cells from human squamous carcinoma bind AG73 and signal through syndecan-1 and integrins resulting in increased motility, invasion, and secretion of MMP9.
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Acknowledgments
This investigation was supported by The State of São Paulo Research Foundation (FAPESP grants 2006/57079-4 and 2008/57103-8) and Brazilian National Council for Scientific and Technological Development (CNPq grants 471751/2003-0, 304868/2006-0 and 470622/2007-5). Adriane S. Siqueira and Letícia N. Gama-de-Souza are recipients of Graduate fellowships from FAPESP (2007/51950-8 and 2005/55602-9). João J. V. Pinheiro is recipient of a Post Doctoral fellowship from CNPq (504667/2008-4).
Conflict of Interest Statement
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Siqueira, A.S., Gama-de-Souza, L.N., Arnaud, M.V.C. et al. Laminin-derived peptide AG73 regulates migration, invasion, and protease activity of human oral squamous cell carcinoma cells through syndecan-1 and β1 integrin. Tumor Biol. 31, 46–58 (2010). https://doi.org/10.1007/s13277-009-0008-x
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DOI: https://doi.org/10.1007/s13277-009-0008-x