Cancer Gene Therapy (2016) 00, 1–10 © 2016 Nature America, Inc., part of Springer Nature. All rights reserved 0929-1903/16 www.nature.com/cgt

ORIGINAL ARTICLE

S100A7 has an oncogenic role in oral squamous cell carcinoma by activating p38/MAPK and RAB2A signaling pathway KK Dey1,5, R Bharti1, G Dey1, I Pal1, Y Rajesh1, S Chavan2, S Das1, CK Das1, BC Jena1, P Halder1, JG Ray3, I Kulavi4 and M Mandal1 Oral cancer consists of squamous cell carcinoma within the oral cavity or on the lip. The clinical prognosis of this cancer is mostly poor owing to delayed diagnosis and a lack of appropriate early detection biomarkers to identify the disease. In the current study, we investigated the role of the S100A7 calcium-binding protein in oral squamous cell carcinoma as an activator of the p38/MAPK and RAB2A signaling pathway. The aim of the present study was to determine whether S100A7 and RAB2A have a role in tumor progression and to assess their potential as early detection biomarkers for oral cancer. This study elucidated the functional and molecular mechanisms of S100A7 and RAB2A activity in oral cancer, leading us to conclude that S100A7 is the major contributing factor in the occurrence of oral cancer and promotes local tumor progression by activating the MAPK signaling pathway via the RAB2A pathway. We hypothesize that S100A7 affects cell motility and invasion by regulating the RAB2A-associated MAPK signaling cascades. Also, the downregulation of S100A7 expression by RNA interference-mediated silencing inhibits oral cancer cell growth, migration and invasion. Cancer Gene Therapy advance online publication, 21 October 2016; doi:10.1038/cgt.2016.43

INTRODUCTION Head and neck cancer is the sixth most common cancer worldwide. Most oral cancers consist of oral squamous cell carcinoma (OSCC) within the oral cavity or on the lip.1 The development of oral cancer is related to a multistep series of biological events, and the key risk factors in developing OSCC are tobacco use, alcohol consumption,2 the use of tobacco-free smoking products, genetic mutation and human papilloma virus infections.3–5 Worldwide, around 600 000 people are diagnosed with head and neck cancer each year. Of these, 40% survive for o5 years after the initial detection of disease progression.6,7 Although cigarette smoking is the most common form of tobacco use worldwide, the use of smokeless tobacco, or chew, is associated with oral cavity cancers. The clinical outlook for oral cancer is generally poor, owing to delays in its detection and the lack of molecular markers for the progress of the disease. More than 70% of all cancers in India are found when the disease is so advanced that treatment is much less effective than would have been the case if the cancer had been detected at an early stage.8 Therefore, it is vital to identify early detection biomarkers for oral cancer in order to facilitate early diagnosis and improve the survival rate. On the basis of earlier findings obtained with a mass spectrometry–based proteomics approach, we have identified two novel signature proteins, RAB2A and S100A7,9 and further elucidated their role in oral cancer progression. S100A7 (psoriasin) belongs to the S100 gene family10 and was first isolated from psoriasis-affected skin.11 It is an 11.4-kDa secretory protein that is often responsible for inflammatory responses in the skin.12–14 Furthermore, S100A7 expression was observed due to altered keratinocyte differentiation in the skin,15,16 and

the differential expression of S100A7 was noted in squamous cell cancer of the bladder17 and in breast carcinoma.18,19 Intensive studies revealed that increasing expression of S100A7 was closely associated with the early development of oncogenesis. S100A7 was upregulated in ductal carcinoma, but its expression was relatively low in adjacent invasive carcinoma.19 Distinct changes in the expression profile of S100A7 were also noticed in benign tumors and in high-grade ductal carcinoma in situ.19,20 Altered expression of S100A7 also appeared to be associated with poor prognosis in several invasive tumors, predominantly in ERα- and ERβ-negative tumors.21 Previous studies suggested that S100A7 expression is generally limited to the epithelial part of the skin, breast and bladder.12,17,21–23 An earlier report revealed that the functional activity of S100A7 is regulated by a small cluster of genes, and serial analysis of gene expression profiling demonstrated that this gene population is significantly upregulated in actinic keratosis, as compared with normal skin.14,15 Factors regulating S100A7 include ultraviolet radiation, calcium and altered cell attachment to the skin24–28 along with various physiological factors such as confluency, growth factor deprivation and the loss of cellular attachment in breast epithelial cells.18,20 Cell stress due to ultraviolet radiation promotes the activation of the AP-1 transcription factor complex mediated by the c-Jun/JNK pathway,29 whereas skin tumorigenesis, progression and invasion are caused by the stimulation of AP-1 pathway.30 Although S100A7 has been reported to have a role in squamous cell cancer, the molecular mechanisms of its effects are not well understood. However, there are reports that the S100A7 gene, together with nuclear factor kappa-B (NF-κB) transcription factors, has a role in inducing cell growth by stimulating proliferation

1 School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, India; 2Manipal University, Mangalore, Karnataka, India; 3Dr Rafi Ahmed Dental College and Hospital, Kolkata, West Bengal, India and 4Bankura Sammilani Medical College, Bankura, West Bengal, India. Correspondence: Professor M Mandal, School of Medical Science and Technology, Indian Institute Of Technology, Kharagpur 721302, India or Dr KK Dey, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105-3678, USA E-mail: [email protected] or [email protected] 5 Current address: St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105-3678, USA. Received 27 July 2016; revised 28 August 2016; accepted 30 August 2016

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genes.31,32 S100A7-c-Jun activation domain binding protein 1 pathway develops pro-survival pathways in breast cancer.33 The upregulation of S100A7 expression triggers enhanced tumorigenicity and the anchorage-independent growth of cancer cells through Akt phosphorylation, leading to the development of anoikis resistance in head and neck cancer cells.14 S100A7 has a key role in the progression of the skin cancer GATA-3/caspase-14 pathway.34 RAGE, a candidate biomarker for cancer, has a functional role in RAGE/S100A7 signaling, connecting inflammation to aggressive breast cancer growth.35 S100A7 also increases the expression of ROS and vascular endothelial growth factor and promotes endothelial cell proliferation via RAGE mediation,36 and estrogen receptor β regulates S100A7 in human breast cancer development.37 Earlier data indicated that S100A7 expression was transcriptionally downregulated by interferon-γ, mediated by the activation of the STAT1 signaling pathway. The increased viability of S100A7expressing cells after interferon-γ exposure leads to the development of apoptosis-resistant cancerous cells.38 Studies also suggest that inflammatory cytokines can regulate S100A7 expression in cancer,39 that S100A7 has an important role in tumor formation by the Jak/STAT3 signaling pathway,40 and that the tumorsuppressive effects of S100A7 are also mediated through the β-catenin/T-cell factor 4 (TCF4) protein pathway.41 Another protein, ras-related protein 14 (RAB14), shows significant fold change and is overexpressed in non-small-cell lung carcinoma.42 RAB proteins are small-molecular-weight membranebound guanosine triphosphatases (GTPases). RAB14 has highly conserved domains that are responsible for GTP binding and hydrolysis, and it is also involved in vesicular fusion and trafficking. This protein is derived from pre-Golgi intermediates, and its main task is to transport protein from the endoplasmic reticulum to the Golgi complex.43 It is crucial for vesicular transport from the Golgi to the nuclear envelope in spermatids during acrosomal biogenesis.44 RAB14 is a membranous protein, which is involved in organelle biogenesis and vesicular fusion; malfunctioning of the RAB protein leads to cancer progression. In the present study, we wished to explore the oncogenic role of S100A7, an early detection biomarker for OSCC, by activating the p38 mitogen-activated protein kinase (MAPK) and RAB2A signaling pathway. RAB2A was found to be localized in the cytoplasm, whereas S100A7 was expressed in both the cytoplasm and membrane within the cancerous tissue. The changes in the expression levels of relevant proteins were further studied after silencing the S100A7 gene. Quantitative reverse transcription (RT)PCR and knock-down analysis in FaDu cells suggested that the downregulation of the RAB2A gene occurs via the p38 MAPK signaling pathway. RNA interference (RNAi)-mediated knock down of S100A7 inhibited cell growth, proliferation, migration and invasion in vitro, and by altering the expression of epithelial-tomesenchymal transition (EMT) regulatory proteins and matrix metalloproteinase (MMPs) and inducing apoptosis.

Technology, Danvers, MA, USA, cat no: 2872), BAX (Cell Signaling cat no: 2772), anti- rabbit PARP (Cell Signaling cat no: 9542), XIAP (Cell Signaling cat no: 2042), ATF-2 (Cell Signaling cat no: 9222), Pan Ras (Santa Cruz cat no: S-32), K-ras (Santa Cruz cat no: S-30), p38 MAPK (Cell Signaling cat no: 9212), Myc (Cell Signaling cat no: 9402), p53 (Cell Signaling cat no: 9282), NF-κB p65 (Santa Cruz cat no: sc-372), Vimentin, N-cadherin, Snail and E-cadherin (Cell Signaling cat no: 9782), mouse monoclonal anti–procaspase 3 (Santa Cruz cat no: sc-7148); mouse monoclonal anti–β-actin (Sigma-Aldrich cat no: A5316-100UL, St Louis, MO, USA); and horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology cat no: sc-2030) and goat anti-mouse IgG (Santa Cruz Biotechnology cat no: sc-2005). Alexa flour-conjugated anti-rabbit IgG and FITC-conjugated antimouse IgG were purchased from Life Technologies (Thermo-Fisher Scientific cat no: A-21428, cat no: 62-6511 Waltham, MA, USA). Chemiluminescent peroxidase substrate, methylthiazoletetrazolium (MTT) and RNase A reagents were purchased from Sigma-Aldrich.

RNA extraction, cDNA synthesis, reverse transcription PCR and quantitative real-time PCR The isolation of total RNA from cells grown in culture and tissue was carried out using an RNeasy Mini Kit (Qiagen, Germantown, MD, USA). The cDNA was then synthesized using SuperScript II RNase H − Reverse Transcriptase (Invitrogen, Lidingö, Sweden). This was followed by RT-PCR amplification in a 25-μl reaction volume, as the method was standardized.45 The PCR amplification consisted of an initial incubation at 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 54°C for 45 s and 72 °C for 60 s; and a final extension at 72 °C for 10 min.46 The amplified products were separated by electrophoresis on 1.2% agarose gels, which were stained with ethidium bromide then photographed and analyzed using the Gel Doc imaging system (Bio-Rad, Hercules, CA, USA).47 Semi quantitative analysis was performed by comparing the results for S100A7 and RAB2A mRNA with those for GAPDH. For relative expression analysis, real-time PCR and a LightCycler instrument (Roche Applied Science, Bromma, Sweden), together with the LightCycler FastStart DNA Master SYBR Green I kit (Roche Applied Science) were used. The sense and antisense primers for the S100A7, RAB2A and GAPDH genes were as follows: for S100A7, the forward primer was 5′-CACCAGACG TGATGACAA-3′ and reverse primer was 5′-GGCTATGTCTCCCAGCAA-3′; for RABA2, the forward primer was 5′-TCCACCAGGGTCTGATTTTT-3′ and the reverse primer was 5′-TTGAAGCGGAGAAGGAGACG-3′ and for GAPDH the forward primer was 5′-AGCAACAGGGTGGTGGAC-3′ and the reverse primer was 5′-GTGTGGTGGGGGACTGAG-3′. An aliquot of 2 μl cDNA was added to 18 μl PCR Master Mix (3 mM MgCl2, 0.5 mM primers (for S100A7, RAB2A and GAPDH)) and 2 μl of LightCycler FastStart DNA Master SYBR Green. The melting curves were analyzed between 60 and 95 °C at intervals of 0.5 °C, with each temperature held for 20 s, and internal calibration curves were plotted by the real-time software. The melting curve peak and cycle number (Ct) with signals crossing a threshold in the logarithmic phase were recorded, and the relative levels of gene expression were calculated by the 2-ΔΔCt method.48 Finally, the relative expression levels were derived by normalizing the Ct values of target genes to an endogenous internal reference (GAPDH) and a calibrator (control cells).

Protein isolation and western blotting

The FaDu human oral cancer cell line was obtained from the National Centre for Cell Science (Pune, India). The cells were incubated at 37 °C in a 5% CO2 atmosphere with 95% humidity in Minimum Essential Medium (MEM) (Gibco-BRL, Rockville, MD, USA) supplemented with 10% heat inactivated fetal bovine serum (Gibco-BRL).

The FaDu cells were grown in cell culture dishes and transfected with S100A7 siRNA or control treatment for 48 h. Protein was isolated from the cells by treating them with NP-40 lysis buffer (Sigma-Aldrich) for 60 min at 4 °C. The proteins were separated by SDS–PAGE and transferred to nitrocellulose membranes. The blots were blocked with phosphatebuffered saline Tween containing 3% bovine serum albumin for 1 h and subsequently incubated with the primary antibodies overnight at 4 °C. The blots were washed then incubated with secondary antibodies, and the proteins were visualized with a chemiluminescence kit and an ImageQuant LAS 4000 biomolecular imaging system (GE Healthcare BioSciences AB, Uppsala, Sweden). Densitometric analysis was performed with GelQuant software (BiochemLabSolutions.com).14

Reagents

Western blot analysis of human tissue

The small interfering RNA (siRNA) of S100A7 was a gift from Dr Peter H Watson, University of Manitoba, and Winnipeg, Manitoba, Canada. For immunoblot analysis, the following antibodies were used: anti-RAB2A (Santa Cruz Biotechnology sc-26547, Santa Cruz, CA, USA); anti-S100A7 (Imgenex cat no: NBP2-24911, San Diego, CA, USA); Bcl-2 (Cell Signaling

Human oral tissue specimens (n = 4) were collected from the Department of Dentistry, Bankura Sammilani Medical College, Bankura, West Bengal, with the approval of the Institutional Ethical Committee of Bankura Sammilani Medical College (approval no. PR-HC/6-119/2895(8)). All patients agreed to participate in this study and provided signed, written informed

MATERIALS AND METHODS Cell lines

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3 consent. Tissue samples were homogenized in tissue lysis buffer (SigmaAldrich), and the proteins were isolated and examined by western blot analysis.14,49

Immunoprecipitation Cells treated with siRNA were harvested and lysed with NP-40 cell lysis buffer (Sigma-Aldrich). The supernatant was subjected to immunoprecipitation by using the Dynabeads Protein A Immunoprecipitation Kit (Novex, Life Technologies AS, Oslo, Norway) with anti-RAB2A antibody and co-immunoprecipitation of S100A7 protein was then carried out by western blot.

Transient transfection For studies of invasion, migration, proliferation and morphology, cells were transfected with siRNA control vector and siRNAs against S100A7 (Qiagen, Mississauga, ON, Canada) and RAB2A (Santa Cruz Biotechnology, Santa Cruz). FaDU cells were seeded at 1.5 × 105 cells per well and allowed to grow to roughly 70% confluence on the day of transfection. Cells were transfected with Dharmafect-4 (Thermo Fisher Scientific, Waltham, MA, USA) and 50–100 nM siRNA in OptiMEM reduced serum medium (Invitrogen, Life Technologies, Carlsbad, CA, USA). The siRNA constructs used here were as follows: (1) siRNA control, (2) siRNA against S100A7 and (3) siRNA against RAB2A. After transfection, the cells were incubated for 16–24 h and further experiments were performed.

Cell proliferation and morphological studies Cell proliferation was determined by a MTT assay, as described previously.50 Briefly, 5000 cells per well were seeded in 12-well plates and incubated at 37 °C for 24 h. After siRNA treatment, cells were incubated at 37 °C for 48 h, then 200 μl sterile MTT dye (1 mg ml − 1, Sigma, St. Louis, MO, USA) was added. After incubation for 4 h at 37 °C in 5% CO2, the MTT medium mixture was removed and 200 μl dimethyl sulfoxide was added to each well, as described earlier. Each assay was performed in triplicate, and the percentages of cell viability were determined by measuring the absorbance at 570 nm (OD570) for treated cells and control cells. For cell morphology studies, FaDu cells were grown and transfected on glass slides. The slides were then washed and fixed in 10% formaldehyde, and the images were captured by phase-contrast microscopy. All experiments were carried out in triplicate.

Wound-healing assay The effect of siRNA on cell migration was determined by plating FaDu cells (1 × 104) in 6-well plates with complete growth medium.51 After 24 h, a scratch was made through each cell monolayer with a 200-μl pipette tip, and the cells were treated with S100A7 siRNA or control, both in 3 ml of complete medium. At 48 h post treatment, the cells were stained with eosin and hematoxylin, and the scratches in the monolayers were observed under an inverted phase-contrast microscope with a × 20 objective (Leica DMR, Germany). The wounds were photographed (with a × 4 objective) at the 0- and 48-h time points. The healing was quantified by measuring the minimum distance in millimeters between the edges of the scratch in each monolayer with Adobe Photoshop (v.9.0.1; Adobe Systems, San Jose, CA, USA). The experiment was performed three times.

Gelatin zymography Supernatants from FaDu cells (5 × 104 cells per well) transfected with S100A7 siRNA for 48 h were collected for analysis of the MMP activity by SDS–PAGE under non-reducing conditions, as described previously.52 The densitometry analysis of the stained bands was performed using Image Master 2D Platinum 7.0 Software (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).

Capillary-like tube formation (HUVEC) assay Human umbilical vein endothelial cells (HUVECs) were seeded at ~ 20 000 cells per well on polymerized matrigel with or without 35 ng ml − 1 vascular endothelial growth factor in the presence of various conditioned media. After incubation for the specified time, the conditioned medium from cells treated with S100A7 siRNA was collected and used for tube formation assay, as described previously.53

Statistical analysis All statistical analysis was performed using Graph Pad Prism 5 software (La Jolla, CA, USA). Statistical significance was assessed by one-way analysis of variance. P-values of o0.05 were considered significant.

RESULTS Expression of S100A7 and RAB2A in oral cancer tissue and cancer cell lines We examined the expression of RAB2A and S100A7 in oral cancer tumor tissue, normal oral tissues, FaDu oral cancer cells and normal oral keratinocytes, primarily by using western blotting. Our results showed that the expression of S100A7 and RAB2A was substantially higher in oral cancer tissues than in the adjacent normal oral tissues (Figures 1a and b). Western blot analysis also confirmed that S100A7 and RAB2A protein expression was consistently higher in the FaDu cell line than in the normal oral keratinocytes. Next, we semi quantitatively analyzed the expression levels of S100A7 and RAB2A mRNA in oral cancer tumor tissue, normal oral tissues, FaDu oral cancer cells and normal oral keratinocytes by RT-PCR. The mRNA expression in both the cancer tissue and the FaDu cells was higher than in the normal control tissue and normal cell line (Figures 1c and d). These results suggested that both S100A7 and RAB2A were overexpressed and upregulated in oral cancer. From this, we predicted that both genes played a key role in oral cancer tumor progression, prompting further analysis.

Migration and invasion assay

Cellular localization of RAB2A and S100A7 The S100A7 and RAB2A proteins showed aberrant expression in OSCC tissue and cells, and this finding was further validated by RTPCR and western blot analysis. Positive expression of S100A7 at the transcriptional and translational level in FaDu cells met the requirements for an in vitro research tool. In addition, our coimmunoprecipitation and immunofluorescence analysis elaborated a plausible interaction between S100A7 and RAB2A in the membrane. An immunoprecipitation study of protein–protein interaction showed an interaction between S100A7 and RAB2A (Figures 1e and f).

After FaDu cells had been transfected with S100A7 siRNA or empty vector for 24 h, they were seeded in the upper chambers of Corning BioCoat Matrigel Invasion Chambers (Corning, NY, USA) containing 8-μm filters, then placed in Boyden chambers. Conditioned medium containing vascular endothelial growth factor as a source of chemoattractant was placed in the lower compartment of each Boyden chamber. After incubation for 16 h at 37 °C, the cells were fixed with 4% formaldehyde then permeabilized with 100% methanol for 15 min. Micrographs were taken with a bright-field microscope. The results were expressed as the percentage of migrated cells in the treated cells compared with that in the control (untreated) cells. The invasion assay was also carried out using the same procedure with a coated invasion chamber covered with 100 μl of matrigel matrix (0.5 mg ml − 1) (BD Biosciences, Bedford, MA, USA).

RNAi-mediated knock down of S100A7 suppresses oral cancer cell proliferation and morphology in vitro As revealed by an MTT assay, S100A7 knock down considerably reduced the proliferation of FaDu oral cancer cells.50 As shown in Figures 2c and d, FaDu cells transfected with S100A7 siRNA showed significantly reduced proliferation when compared with a non-transfected cancer cell line. There was an apparent decrease in the size, morphology and number of cells that were transfected with S100A7 siRNA, as compared with the control cells. Compared with control cells, transfected cells showed significant morphological changes, reduced cellular extension and shrinkage. This

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4 indicated that silencing affected the morphology of transfected FaDu cells. These results signify a positive relationship between S100A7 expression and the rate of growth and proliferation of oral cancer cells. Knock down of S100A7 expression decreases RAB2A by RNAi-mediated interference in FaDu cells To examine whether S100A7 was related to the progression of cancer cell invasion and metastasis in vitro, FaDu cells were transfected with a siRNA targeting the S100A7 gene. To study the effect of blocking S100A7 expression by RNAi-mediated silencing, western blot and real-time PCR analyses were performed in FaDu cells. As shown in Figures 2a and b, S100A7 protein expression was reduced by 70% after siRNA transfection in FaDu cells, but the control treatment did not alter S100A7 expression. RAB2A protein expression diminished to barely detectable levels in the FaDu cells transfected with S100A7 siRNA, as compared with levels in untransfected cells and those transfected with control siRNA. The effect of S100A7 siRNA on the expression of S100A7 and RAB2A mRNA in FaDu cells was assessed by quantitative real-time PCR. This quantitative approach confirmed that RAB2A expression was decreased by RNAi-mediated gene-specific silencing of the S100A7 gene. These data suggested that downregulation of S100A7 also reduces RAB2A gene expression, which further suppresses the proliferation of oral cancer cells (Figure 2e). S100A7 siRNA induces alteration in pro- and anti-apoptotic proteins We established the chronology of the signaling events after siRNA treatment by employing immunoblotting procedures to examine the mechanistic apoptotic effects of knock down in FaDu cells. The subsequent knock down of FaDu cells for 48 h showed that the

pro-apoptotic BAX protein was upregulated and the antiapoptotic Bcl-2, MCL1 and XAIP proteins were downregulated in transfected cells, as compared to controls, with the regulatory effects progressing over time. Overall, the BAX/Bcl-2 ratio and p53 level were increased by the siRNA treatment, suggesting that this is one mechanism of siRNA-induced apoptosis. We observed cleavage of poly (ADP-ribose) polymerase (PARP) in FaDu cells treated with siRNA (Figures 3a and b). The cleavage was seen in the transfected cells, as there was increased expression of the 85-kDa fragment (cleaved PARP), compared with that in control cells, whereas the 116-kDa protein (uncleaved PARP) was almost absent. Effect of knock down of S100A7 on migration and invasion of oral cancer cells To establish the inhibitory effect of siRNA on oral cancer metastasis, we studied the migration potential of FaDu cells by using a wound-healing assay. We used a pipette tip to create a wound in a confluent cell monolayer and assessed the potential of the cells to migrate and fill up the wound. After 48 h, the cleared region of the wound in monolayers of non-transfected FaDu cells was almost completely filled, whereas there was less migration of transfected FaDu cells (Figures 4a and b). The ability of siRNA to decrease the invasiveness of FaDu cells was further studied by a Boyden chamber assay. Both transfected and control cells were plated for 24 h in the upper chamber, and the number of cells that moved to the underside of the coated membrane was counted 12 h later using a light microscope. The chambers were stained with hematoxylin and eosin and analyzed by photography (Figures 4c and d). From the results, we concluded that knock down of the S100A7 gene in FaDu cells substantially decreased their invasiveness and metastatic potential. These results suggest that S100A7 inhibits the invasiveness of tumorigenic cells.

Figure 1. Analysis of S100A7 and RAB2A expression in normal oral tissues and cells and in squamous cell carcinoma (OSCC) tissues and cells. (a, c) The expression of S100A7 and RAB2A in normal oral tissue, normal oral keratinocytes, oral cancer tissue and the FaDu oral cancer cell line was evaluated using semi quantitative reverse transcription (RT)-PCR and western blot analysis with GAPDH and β-actin as the respective loading controls. (b, d) The intensity of each band was quantified by densitometry scanning, and the relative intensity of each band compared with the corresponding loading control was determined. The results represent the mean ± s.d. for three determinations. Comparisons were made against the untreated samples (U). *P o0.05; **Po 0.01; ***P o0.001. (e) Cellular localization of RAB2A and S100A7 by immunofluorescence. (f) Results of a protein–protein interaction study of S100A7 and RAB2A conducted by immunoprecipitation. NS, not significant. Cancer Gene Therapy (2016), 1 – 10

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Figure 2. Knock down of S100A7 expression decreases RAB2A expression by RNA interference (RNAi)-mediated interference in FaDu cells. (a, b) Expression of S100A7 and RAB2A protein in FaDu cells was significantly reduced by small interfering RNA (siRNA) targeting S100A7, as compared with the untreated (mock) scrambled siRNA. Densitometry analysis was performed to analyze the relative protein levels normalized to β-actin levels. (c) S100A7 knock down significantly inhibited the growth of oral cancer cells, as revealed by an methylthiazoletetrazolium (MTT) assay points, average ± s.d. of different experiments each performed in triplicate o0.05. (d) Phase-contrast micrographs showing the effect of S100A7 knockdown on oral cancer cell morphology; the treated cells exhibit prominent morphological changes (specifically, reduced cellular extension and cell shrinkage) compared with untreated cells. (e) Effect of S100A7 siRNA on S100A7 and RAB2A mRNA expression in FaDu cells, as measured by quantitative real-time PCR. Gene expression was normalized with respect to GAPDH. The results represent the mean ± s.d. of three determinations. Comparisons were made against the untreated samples (U). *Po0.05; **P o0.01; ***P o0.001; NS, not significant.

Figure 3. Small interfering RNA (siRNA) S100A7 induces pro- and anti-apoptotic signaling pathways. (a) Western blot analysis of FaDu cells transfected with siRNA for 48 h. The blot was probed with antibodies to PARP, BAX, Bcl-2, XIAP, MCL1 and pro-caspase 3. (b) The histogram represents the average pixel density of the detected protein expression in three independent experiments. β-actin protein expression was used as a loading control. The results represent the mean ± s.d. of three determinations. Comparisons were made against the untreated samples (U). *Po0.05; **P o0.01; ***P o0.001; NS, not significant.

Knock down of S100A7 inhibits activation of MMP-2 and MMP-9 in oral cancer Significant levels of MMP-2 and MMP-9 secretion have been reported in metastatic oral cancer and are associated with the degradation of the extracellular matrix, a crucial step in metastasis.54 This process has a vital role in the tumor microenvironment by enhancing cancer angiogenesis, cell motility and © 2016 Nature America, Inc., part of Springer Nature.

cancer growth.55 Gelatin zymography analysis showed that siRNAbased silencing inhibited MMP-2 and MMP-9 activity in FaDu cells (Figures 4e and f). Therefore, apart from its anti-vascular endothelial growth factor effect in inhibiting tumor cells, this silencing treatment can inhibit the metastasis and spread of oral cancer cells by reducing the MMP-2/9 levels. In addition, siRNA-based approach increased the anti-metastatic potential by more than twoCancer Gene Therapy (2016), 1 – 10

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Figure 4. Downregulation of S100A7 inhibits cell invasion and the migration potential of FaDu oral cancer cells. (a) In vitro wound-healing assay. Confluent monolayer of FaDu cells was scratched with a 1-ml pipette tip and transfected with gene-specific small interfering RNA (siRNA) and control scrambled siRNA. After 48 h of transfection, the wound was photographed under a microscope. Representative images show cells migrating into the wounded area at 0 and 48 h. (b) The wound areas in monolayers of control and transfected cells were quantified with Adobe Photoshop software. The results are expressed as the mean ± s.e. of three independent experiments. *P o0.05, (compared with the 0 h control). (c) Transfected cells were seeded in a transwell top chamber with or without matrigel and maintained for 24 h at 37 °C. Brightfield photographs of the migrated cells on the lower surface of the transwell chamber were captured. (d) A graphical representation calculated from the percentage of chemoinvasion. (e) The gelatinolytic activity of MMP-2/9 was downregulated by transfection with S100A7 siRNA. (g) Western blot analysis of MMP-2/9 protein expression in transfected cells. (f, h) Densitometry analysis of MMP-2 and MMP-9 protein levels in gelatin and western blots, respectively. The data are the means ± s.e. of three independent experiments. P o0.05 (t-test).

Figure 5. Knock down by small interfering RNA (siRNA) S100A7 inhibits angiogenesis. (a) Inhibition of capillary-like tube formation in vitro (HUVECs assay). Tube formation was observed after 24 h and images were acquired (×10 magnification). The number of capillary-like structures or the tube length in a capillary-like tube formation assay was counted using light microscopy. (b) Graphical representation of the quantification in HUVEC assays; ***P o0.001 (compared with control by unpaired t-test). HUVEC, human umbilical vein endothelial cells.

fold in comparison with the control. Our data demonstrated that knock down of S100A7 inhibited the invasion and migration of cancer cells. Therefore, we next examined the expression level of MMP-2/9 in FaDu cells by using western blot analysis (Figures 4g and h). MMP-2/9 expression was considerably reduced in transfected FaDu cells, as compared with untreated cells. These data suggest that the knock down of S100A7 suppresses the Cancer Gene Therapy (2016), 1 – 10

metastatic potential of oral cancer by initializing MMP-2/9 transcriptional activity. siRNA S100A7 inhibits tube-like capillary formation in vitro In this experiment, HUVECs were grown for use in a tube-like capillary formation assay, which is a widely used in vitro assay for © 2016 Nature America, Inc., part of Springer Nature.

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7 angiogenesis. After 24 h, HUVECs treated only with phosphatebuffered saline had grown rapidly and formed hollow, tube-like structures, whereas HUVECs treated with conditioned medium from siRNA-transfected FaDu cells showed considerably reduced tube formation compared with controls (Figure 5). This result suggested that the knock down of S100A7 inhibited HUVEC differentiation into tube-like structures during angiogenesis. Knock down of siRNA S100A7 inhibits EMT in vitro in oral carcinoma Cancerous cells acquire increased metastatic potential by gaining characteristics of a mesenchymal-like state. The development of metastatic cascade stimulates EMT, which endows the cancer cells with increased invasive and migratory potential and leads to an adverse prognosis in different types of cancer, including oral cancer. Interestingly, we found that transient transfection of S100A7 siRNA into FaDu cells decreased S100A7 expression and increased the cell membrane assembly of E-cadherin, as shown by immunofluorescent staining (Figure 6a), suggesting that downregulating S100A7 not only increased E-cadherin signaling but also inhibited E-cadherin–mediated cell adhesion. Western blot analysis further confirmed that the level of E-cadherin expression was higher in cells transfected with S100A7 siRNA than in cells transfected with control siRNA. In light of these observations, we further explored the possible mechanisms of E-cadherin activity by downregulating S100A7, and we subsequently examined the expression of

mesenchymal markers such as N-cadherin, snail and vimentin. These markers are downregulated by the knock down of S100A7 protein in FaDu oral cancer cells (Figures 6b and c). Taken together, these results indicate that the downregulation of S100A7 enhances E-cadherin signaling, that S100A7 is required for the efficient assembly of E-cadherin at the cell membrane, and that the downregulation of S100A7 impairs E-cadherin–mediated cell-cell adhesion. Gene silencing of S100A7 suppresses p38 MAPK and RAB2A signaling in oral cancer The p38 MAPK signaling pathway participates in the different steps of cancer progression and metastasis. The p38 MAPK subfamily is associated with the MAPK family that is triggered by inflammatory cytokines and a variety of environmental factors. P38 MAPK activity has an important role in tumor progression and also mediates various biological processes. Tumorigenesis is a multistep complex process that involves the cooperation of many genes and, particularly, the activation of oncogenes and inactivation of tumor suppressor genes. Furthermore, knock down of S100A7 caused the induction of apoptosis, followed by downregulation of the RAB2A-mediated p38 MAPK pathway. As a result of the blockade of S100A7 with RNAi silencing the downstream effector proteins, such as ras family proteins and p38 MAPK, are significantly downregulated relative to their levels in controls. S100A7 siRNA interferes with the expression of p38 at the protein

Figure 6. Effect of small interfering RNA (siRNA) S100A7 on epithelial-to-mesenchymal transition (EMT)-associated marker in FaDu cells. (a) Immunofluorescence study of cellular localization of E-cadherin and S100A7. (b) FaDu cells transfected with siRNA were subjected to western blot and densitometry analysis to determine the relative amounts of the EMT-related markers E-cadherin, N-cadherin, snail and vimentin. β-actin was used as a loading control in all western blots. The results are representative of at least three independent experiments. (c) Graphical representation of the densitometry analysis of the levels of EMT markers in cell lines. Data were plotted as means ± s.e. *Po 0.05; (compared with the control group). © 2016 Nature America, Inc., part of Springer Nature.

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8 level in FaDu cells, resulting in the inhibition of cell proliferation. Furthermore, p38 MAPK is involved in regulating several transcription factors, including ATF-2, Stat1, p53, Myc and (indirectly) NF-κB p65, via the activation of MSK1/2, whereas p53 shows increased expression with respect to controls (Figure 7). S100A7 may regulate RAB2A gene expression through MAPK activation in oral cancer cells and may be a potential target for treating oral cancer.

DISCUSSION In cancer patients, genetic mutations can lead to tumor formation. In addition, these mutations are transcribed and translated into proteins with increased-, decreased- or even complete loss of function.56 In modern cancer research there is a strong demand for new, efficient protein markers and therapeutic targets, which might be a post-transcribed or translated product that can be used as an early detection marker. S100A7 (psoriasin) has a pivotal

Figure 7. S100A7 modulates the p38/mitogen-activated protein kinase (MAPK) signaling pathway via RAB2A (ras-related protein) in oral cancer. (a) Western blot analysis of FaDu cells transfected with small interfering RNA (siRNA) S100A7 for 48 h. The blots were probed with antibodies to S100A7, RAB2A, Pan ras, K-ras, p38 MAPK, Myc, ATF-2, p53 and NF-κB p65. (b) Histogram representing the average pixel density of the expression of three independent experiments. β-actin was used as an internal probe for equal loading. Data were plotted as means ± s.e., *Po0.05, **P o0.01 and ***P o0.001 (compared with the control group).

Figure 8. Schematic representation of the contribution of S100A7 to oral squamous cell carcinoma (OSCC) by activating the p38/mitogenactivated protein kinase (MAPK) and RAB2A signaling pathways. Cancer Gene Therapy (2016), 1 – 10

© 2016 Nature America, Inc., part of Springer Nature.

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role in the malignant transformation in several cancers14 and could be a candidate diagnostic marker or therapeutic target. S100A7 signaling has an important function in the pathogenesis and progression of human oral cancers,14 but its precise role and mechanism in tumor invasion remains unclear. Previous studies have suggested that, unlike the other category of S100 proteins, S100A7 expression is generally restricted to the epithelial tissue in skin, breast and bladder cancer.12,14,17,21–23 This has already been discussed in a review of the literature on the relation between S100A7, cancer and the various signaling pathways.14 In the current study, we observed the tumor-enhancing effects of S100A7 in FaDu oral cancer cells. We also demonstrated the role of the MAPK/ERK pathway (also known as the Ras/Raf/MEK/ ERK pathway). The signal starts when a signaling molecule (ligand) binds to the receptor on the cell surface and ends when the DNA in the nucleus expresses a protein and produces a change in the cell. P38 MAPKs are activated by inflammatory cytokines and a variety of environmental stresses and have a significant role in tumor progression. Earlier reports based on proteomics data had indicated that S100A7 and RAB2A were both upregulated in oral cancer.9,57 The analysis of S100A7 and RAB2A expression in normal oral and OSCC tissues and cells was further validated by our RT-PCR and western blot analysis (Figure 2a). The genes encoding S100A7 and RAB2A were both upregulated aberrantly in oral cancer, and S100A7 mRNA and protein were positively expressed in FaDu cells. Therefore, FaDu cell lines could be used to model changes in cell biological characteristics after silencing of the S100A7 gene. Transfection with siRNA is a putative preventive treatment for human oral cancer. Our quantitative real-time PCR analysis showed that S100A7 expression was reduced by siRNA transfection, leading to the downregulation of the RAB2A gene via the p38 MAPK signaling pathway. An MTT cell proliferation assay demonstrated that ~ 96% silencing of the gene was achieved by the siRNA targeting S100A7 and RAB2A in FaDu cells. We also investigated the functional role and molecular mechanisms of S100A7 activity in oral cancer. Knock down of S100A7 expression by RNAi inhibits oral cancer cell growth, migration and invasion in vitro. This knock down of S100A7 also suppresses the metastasis of oral cancer cells by lowering the expression of MMP-2 and MMP-9 transcriptional activity. We hypothesized that S100A7 affects cell motility and invasion by regulating the RAB2A RAB associated with the MAPK signaling cascades. We used anti-RAB2A antibody to perform immunoprecipitation with protein extracts from FaDu cells and also analyzed the co-immunoprecipitation of RAB2A with S100A7. Furthermore, an immunofluorescence study also predicted the cellular localization of RAB2A and S100A7 in most oral cancers. The roles of anti- and pro-apoptotic proteins were studied by silencing S100A7 and decreasing the ratio of Bcl2/BAX in FaDU cells. The data strongly supported the hypothesis that S100A7 upregulated the Bcl-2/BAX ratio and, consequently, anti-apoptotic proteins such as Bcl-2, XIAP and MCL1 showed decreased expression in siRNA-transfected FaDu cells. E-cadherin is the foremost tumor suppressor protein. Cancer cells downregulate E-cadherin to allow for translocation in the EMT process.58 This study aimed to elucidate the biological consequences of RNAi silencing on EMT in oral carcinoma cells in vitro. Immunoblot analysis revealed the altered expression of several epithelial and mesenchymal markers, such as E-cadherin, snail, vinculin and vimentin, after treatment with siRNA (Figure 2d). Immunofluorescence studies further revealed the effect of siRNA on the interaction between S100A7 and E-cadherin, which alters the expression of epithelial and mesenchymal markers (Figure 2e). E-cadherin expression has a close relation with S100A7 and maintains coherent function in adheren junctions. As a consequence of the blockade of S100A7 with RNAi silencing, the downstream effector proteins, such as the ras family proteins and p38 MAPK, are significantly downregulated © 2016 Nature America, Inc., part of Springer Nature.

9 by comparison with the control. S100A7 siRNA interferes with p38 expression at the protein level in FaDu cells, resulting in the inhibition of cell proliferation. Furthermore, p38 MAPK is involved in regulating several transcription factors, including ATF-2, Stat1, p53, Myc and (indirectly) NF-κB p65,59 via activation of MSK1/2, whereas p53 shows increased expression relative to that in the control. S100A7 may regulate the expression of the gene encoding RAB2A through MAPK activation in oral cancer cells and may be a potential target for treating oral cancer. A schematic representation of S100A7-mediated action regulating apoptosis, cell invasion and EMT is shown in Figure 8. The overall outcome of this study leads us to conclude that S100A7 is the major contributing factor in mediating oral cancer cells and local tumor progression and its activation modulates the MAPK signaling pathway via the RAB2A pathway. S100A7 and RAB2A might be useful as dual diagnostic markers for the early detection of primary and recurrent squamous cell carcinoma. Downregulating S100A7 could be an effective treatment approach resulting in the inhibition of invasion and growth. Our findings have clearly demonstrated for the first time a potential role for S100A7 as an early detection marker for oral cancer. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS Kaushik Kumar Dey is a recipient of a Research Fellowship from the Indian Council of Medical research (ICMR), (3/2/2/207/2013/NCD-III) Government of India for financial support, India. This study was supported by grants from the Department of Science and Technology (DST: http://www.dst.gov.in/), (SR/SO/BB-58/2008) and Y Rajesh individual research fellowship grant (2013/603) from the Department of Science and Technology (DST)—INSPIRE, India. This study was also partially supported by the Department Of Biotechnology, New Delhi, India (BT/PR2428/MED/12/517/2011, Date 05 March 2013) and Department of Atomic Energy (DAE), Board of Research in Nuclear Sciences (BRNS), BRNS (35/14/05/2015-BRNS/3053, Date 15 July 2015). We thank Keith A Laycock, PhD, ELS, for editorial assistance and Junmin Peng, PhD, Structural Biology Member, in the preparation of the manuscript.

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