ANTICANCER RESEARCH 34: 1873-1884 (2014)

Impact of S100A8 Expression on Kidney Cancer Progression and Molecular Docking Studies for Kidney Cancer Therapeutics

ZEENAT MIRZA1, HANS-JUERGEN SCHULTEN2, HASAN MA FARSI3, JAUDAH A. AL-MAGHRABI4,5, MAMDOOH A. GARI2, ADEEL GA CHAUDHARY1,2, ADEL M. ABUZENADAH1,2, MOHAMMED H. AL-QAHTANI2 and SAJJAD KARIM2

1King Fahd Medical Research Center, and 2Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Saudi Arabia; 3Department of Urology, and 4Department of Pathology, Faculty of Medicine, King Abdulaziz University Hospital, Jeddah, Saudi Arabia; 5Department of Pathology, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia

Abstract. Background/Aim: The proinflammatory calculations. Detailed analysis of bound structures and their S100A8, which is expressed in myeloid cells under physiological binding free energies was carried out for S100A8, its known conditions, is strongly expressed in human cancer tissues. Its partner (), and S100A8−S100A9 complex (). role in tumor cell differentiation and tumor progression is Results: In our microarray experiments, we identified 1,335 largely unclear and virtually unstudied in kidney cancer. In the significantly differentially expressed , including S100A8, present study, we investigated whether S100A8 could be a in kidney cancer using a cut-off of p<0.05 and fold-change of 2. potential anticancer drug target and therapeutic biomarker for Functional analysis of kidney cancer-associated genes showed kidney cancer, and the underlying molecular mechanisms by overexpression of genes involved in cell-cycle progression, exploiting its interaction profile with drugs. Materials and DNA repair, cell death, tumor morphology and tissue Methods: Microarray-based transcriptomics experiments using development. Pathway analysis showed significant disruption Affymetrix HuGene 1.0 ST arrays were applied to renal cell of pathways of atherosclerosis signaling, liver X carcinoma specimens from Saudi patients for identification of receptor/retinoid X receptor (LXR/RXR) activation, notch significant genes associated with kidney cancer. In addition, we signaling, and interleukin-12 (IL-12) signaling. We identified retrieved selected expression data from the National Center for S100A8 as a prospective biomarker for kidney cancer and in Biotechnology Information Expression Omnibus database silico analysis showed that aspirin, celecoxib, dexamethasone for comparative analysis and confirmation of S100A8 and diclofenac binds to S100A8 and may inhibit downstream expression. Ingenuity Pathway Analysis (IPA) was used to signaling in kidney cancer. Conclusion: The present study elucidate significant molecular networks and pathways provides an initial overview of differentially expressed genes in associated with kidney cancer. The probable polar and non- kidney cancer of Saudi Arabian patients using whole-transcript, polar interactions of possible S100A8 inhibitors (aspirin, high-density expression arrays. Our analysis suggests distinct celecoxib, dexamethasone and diclofenac) were examined by transcriptomic signatures, with significantly high levels of performing molecular docking and binding free energy S100A8, and underlying molecular mechanisms contributing to kidney cancer progression. Our docking-based findings shed insight into S100A8 protein as an attractive anticancer target Correspondence to: Dr. Sajjad Karim, Center of Excellence in for therapeutic intervention in kidney cancer. To our knowledge, Genomic Medicine Research, King Abdulaziz University, PO BOX this is the first structure-based docking study for the selected 80216, Jeddah 21589, Kingdom of Saudi Arabia. Tel: +1 96626401000 protein targets using the chosen ligands. ext. 25123, Fax: +1 96626952521, e-mail: [email protected]; [email protected] and Dr. Mohammed H. AlQahtani, Cancer is a group of diseases caused by dysregulation in Director, Center of Excellence in Genomic Medicine Research, King molecular signaling pathways due to over- and underexpression Abdulaziz University, PO BOX 80216, Jeddah 21589, Kingdom of and loss/gain of function mutations of key associated Saudi Arabia. Tel: +1 96626401000 ext. 25962, Fax: +1 96626952521, e-mail: [email protected] with altered cell growth and cell-cycle progression. It develops via a multi-step process of initiation, promotion, and progression Key Words: Kidney cancer, profiling, S100A8, (1, 2). Among all types of urological cancers, kidney cancer is docking, anticancer target, Saudi Arabia. the second leading cause of death in adults, mainly due to lack of

0250-7005/2014 $2.00+.40 1873 ANTICANCER RESEARCH 34: 1873-1884 (2014) promising biomarkers for effective target therapy. We conducted presence and concentration of free calcium molecules. S100A8 transcriptomic profiling and functional pathway analysis to study and S100A9 share strong and can form the role of the S100A8 protein in tumor cells and in cancer heterodimers (without calcium) or heterotetramers (with development. Docking study revealed the potential of S100A8 calcium) (18). Three-dimensional crystallographic analysis as a target of therapeutic importance. reveals that calcium-bound C-terminal EF-hand loops are S100A8 is a low-molecular-weight proinflammatory protein necessary for tetramerization (33). Overall, this indicates that of 10 kDa, belonging to the S100 family of Ca2+-binding the S100A8, S100A9 or S100A8−S100A9 complex could be elongation factor (EF) hand-type proteins constitutively targeted to prevent the tumor cell migration and growth. expressed by myeloid cells, such as neutrophils and activated Molecular docking gives an optimized conformation and monocytes, under physiological conditions (3-6). However, relative orientation for both the protein and ligand molecule increased expression is seen in epithelial cells under pathological such that the free energy of the overall bound system is conditions, including inflammation and wound healing (7). minimal. Non-selective non-steroidal anti-inflammatory drugs S100A8 is an essential gene for life since S100A8 knock-out (NSAID) such as aspirin, diclofenac, indomethacin, ibuprofen mice die during embryonic development (8). Enhanced and naproxen inhibit both cyclooxygenase, COX-1 and COX2 expression of S100A8 is one of the hallmarks of chronic but can lead to drastic side-effects such as gastric ulceration. inflammation and epithelial cancer. Overexpression of S100A8 is However, selective NSAIDs such as celecoxib (Celebrex®) are found in various types of carcinomas, including breast (9-10), much safer and only inhibit COX2 found at sites of prostate (11-12), lung (13), gastric (14), hepatic (15), pancreatic inflammation, more than that which is normally found in the (16) and colorectal (17) cancer. Nevertheless, little research has stomach, blood platelets, and blood vessels (COX1). been carried out on its expression in different types of tumor Aspirin, a prototypical analgesic, is very commonly cells or its correlation with cancer development; the differential administered for the treatment of mild to moderate pain. It expression pattern or role of S100A8 in progression of kidney has anti-inflammatory and anti-pyretic properties and acts as cancer has not been reported as far as we are aware. an inhibitor of cyclooxygenase (both COX1 and COX2), Several lines of evidence point to vital functions of S100A8 which results in the inhibition of prostaglandin biosynthesis. during tumorigenesis and, although its exact role within the It also inhibits platelet aggregation and is used in the tumor environment is still not understood, different tumor- prevention of arterial and venous thrombosis. Daily aspirin promoting effects have been proposed. S100A8 preferentially intake has been shown to be beneficial in treatment of cancer forms heterodimeric complexes with S100A9 and lowering the risk of cancer development (34-37). The (S100A8−S100A9) (18), which undergoes conformational molecular mechanisms involved in anticancer action of changes upon Ca2+ binding and functions as a sensor of aspirin have not yet been elucidated. intracellular Ca2+ (19). Extracellularly, S100A8−S100A9 acts Dexamethasone, is a synthetic adrenocortical steroid (analog as ligand for different receptors, including the receptor for of glucocorticoids) primarily used for its anti-inflammatory advanced glycation end products (RAGE) (10, 20), toll like effects in disorders of many organ systems, and in receptor-4 (TLR4) (21), CD36 receptor (22) and NADPH immunomodulation, as it modifies the body’s immune responses oxidase (23). S100A8 exerts potent pro-inflammatory activity to diverse stimuli. It is an NSAID with anti-pyretic and analgesic (24-26), attracts neutrophils (27), influences myeloid cell actions used primarily in the treatment of chronic arthritic differentiation (28-29), affects transendothelial migration of conditions and certain soft tissue disorders associated with pain phagocytes (30) and induces expression of pro-inflammatory and inflammation. It acts by blocking the synthesis of mediators (31). Studies suggest S100A8 to be an important prostaglandins by inhibiting COX, which converts arachidonic driver of the inflammatory environment, ultimately promoting acid to cyclic endoperoxides, precursors of prostaglandins. cancer progression (10, 15). A recent study showed a role of Inhibition of prostaglandin synthesis accounts for its analgesic, S100A8 at low concentrations in cell growth-promoting activity antipyretic, and platelet-inhibitory actions; other mechanisms by binding to RAGE (9); however, its direct role in tumor cells may also contribute to its anti-inflammatory effects. and tumor progression is ambiguous. It has been demonstrated Diclofenac is another very common NSAID used as an that primary tumors secrete soluble factors, including vascular analgesic, anti-inflammatory and anti-pyretic agent. It is often endothelial growth factor-A (VEGF-A), transforming growth applied to treat chronic pain linked with cancer, in particular factor-beta (TGF-β) and tumor necrosis factor alpha (TNFα), inflammation-associated pain (38). Apart from primarily which induce expression of S100A8 and S100A9 in myeloid inhibiting COX, evidence indicates that it also inhibits and endothelial cells prior to tumor metastasis (13). S100A8 phospholipase A2 and the lipoxygenase pathways, thus reducing also increases the motility of circulating cancer cells by p38 formation of leukotrienes. mitogen activated protein kinase (MAPK)-mediated activation We carried out a series of molecular docking analyses using of tumor cells (32). S100A8 many exist as monomer, aspirin, celecoxib, dexamethasone and diclofenac as inhibitors homodimer, heterodimer or heterotetramer depending on the for S100A8, S100A9, S100A8−S100A9, respectively.

1874 Mirza et al: S100A8: An Anticancer Target for Kidney Cancer

Table I. Gene expression omnibus data series of the expression profile of human kidney cancer.

Accession No Title Sample size Contact Release date

GSE781 Normal and Renal Cell Carcinoma Kidney Tissue, Human 34 Marc E, Lanburg Nov 25, 2003 GSE7023 Renal Cell Carcinoma - Papillary types 1, and 2b, Normal Kidney Tissue 47 Karl Dykema Jun 12, 2007 GSE6344 Gene Expression in Stage 1,2 Normal and Tumor Kidney Cancer 40 John A, Copland Nov 23, 2006

Materials and Methods (Table I). Affymetrix. CEL files were imported to Partek Genomics Suite version 6.6 (Partek Inc., MO, USA). The data were normalized using Patients and samples. The study was performed on samples from random multiple access (RMA) algorithm normalization. Principal patients of the Kingdom of Saudi Arabia, diagnosed with renal cell component analysis (PCA) was performed on all probes to visualize carcinoma. The samples were collected from the King Abdulaziz high-dimensional data with multiple clinicopathological properties. PCA University Hospital, Bakhsh Hospital and King Faisal Specialist was used to assess quality control, as well as overall variance in gene Hospital and Research Center, Jeddah, Sandi Arabia, during 2010-2012. expression between the diseased states. Analysis of variance (ANOVA) Out of 18 received cases, only two passed the criteria to be used for was applied on the complete data set and the list of differentially array expression analysis. One patient was a 61-year-old male, expressed genes was then generated using a false-discovery rate (FDR) diagnosed with clear cell renal cell carcinoma of nuclear grade II and of 0.05 with 2-fold change cut-off. Unsupervised two-dimensional tumor size 4.5×3×4 cm. The other patient was a 47-year-old female, average linkage hierarchical clustering was performed using Spearman’s diagnosed with chromophobic renal cell carcinoma of Fuhrman’s grade correlation as a similarity matrix. II. For gene expression analysis, fresh tumor tissue specimens were obtained from surgical resections adjacent to the sites on which final Functional and pathway analysis. To define biological networks, histological diagnosis was performed. Fresh normal kidney specimens interaction and functional analysis among the differentially regulated were derived from surgically-resected normal kidney tissues. All genes in kidney cancer, pathway analyses were performed using collected tissue specimens were immediately placed in RNALater Ingenuity Pathways Analysis software (IPA) (Ingenuity Systems, (Invitrogen Life Technologies, Grand Island, NY, USA) or RPMI_1640 Redwood City, CA, USA). Statistically differentially expressed datasets medium (GIBCO BRL, Grand Island, NY USA). containing 1,335 genes and their corresponding probesets ID, gene symbol, gene ID as clone identifier, p-value and fold change Ethical approval. All patients included in the study provided written values were uploaded into IPA. The functional/pathway analysis of IPA informed consent. The study was reviewed and approved by the Center identifies the biological functions, diseases and pathways that are most of Excellence in Genomic Medicine Research (CEGMR) Ethical significantly altered for the differentially expressed gene set. The Committee (08-CEGMR-02-ETH). significance of the relation between the expression data and the canonical pathways were calculated by ratio or Fisher’s exact test. RNA extraction and array processing. Total RNA was extracted from freshly-preserved kidney tissue specimens with the Qiagen RNeasy Mini Molecular docking. To investigate the role of S100A8 and its partner, Kit (Qiagen, Hilden, Germany) including an on-column DNAse S100A9, as inflammatory mediators and to establish their involvement treatment according to the manufacturer’s recommendations. Quality of in inflammation-associated cancers at molecular level, we carried out the purified RNA was verified on an Agilent 2100 Bioanalyzer (Agilent docking studies. The molecular structure of aspirin, celecoxib, Technologies, Palo Alto, CA, USA). The mean RNA integrity number dexamethasone and diclofenac were retrieved from PubChem compound (RIN) for processed samples was 8.0 only for two samples. Hence, the database with CID 2244, 2662, 5743 and 3033 respectively (Figure 1). remaining were excluded from further expression studies. RNA Protein targets taken for docking studies were S100A8 homodimer, concentrations were determined using a NanoDrop ND-1000 S100A9 homodimer and S100A8−S100A9 heterodimer complex. The spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). crystal structure of identified cancer signaling target proteins were Two hundred and fifty nanograms of each RNA sample were processed retrieved from (PDBID: 1MR8, 1IRJ, and 4GGF). according to the manufacturer’s recommendations (Life Technology). Structure visualization was profound using PyMol (39) (Figure 2). After fragmentation and labeling, the samples were hybridized at 45˚C Docking calculations were carried-out using Molecular Docking for 17 h to Human Gene 1.0 ST GeneChip arrays (Affymetrix, Santa Server (40). The Merck molecular force field 94 (MMFF94) (41) was Clara, CA, USA). These arrays are conceptually based on the human used for energy minimization of ligand molecules: aspirin, celecoxib, genome sequence assembly UCSC hg18, NCBI Build 36 and dexamethasone and diclofenac. Gasteiger partial charges were added to interrogated with a set of 764,885 probes, 28,869 annotated genes. the ligand atoms. Non-polar hydrogen atoms were merged, and rotatable bonds were defined. Molecular docking of each ligand was performed Gene expression analysis. We conducted expression profiling on a small individually with (S100A8)2 homodimer (1MR8), (S100A9)2 group of samples (two renal cell carcinomas and four normal kidney homodimer (1IRJ, in the presence and absence of prior existing ligand tissues), which requires confirmation by other studies. Therefore, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate, CPS, selected kidney cancer expression data (GSE781, GSE7023, and also known as CHAPS) and (S100A8−S100A9)2 hetrotetramer (4GGF) GSE6344) were retrieved from the gene expression omnibus database protein models to predict the binding orientation and interaction. for comparative meta-analysis and our own experimental findings were Essential hydrogen atoms, Kollman united atom type charges, and verified with a larger independent dataset available in the public domain solvation parameters were added with the aid of AutoDock tools (42).

1875 ANTICANCER RESEARCH 34: 1873-1884 (2014)

Figure 2. Structure visualization of cancer signaling target proteins S100A8 (1MR8), S100A9 (1IRJ) and S100A8−S100A9 (4GGF) retrieved from Protein Data Bank (PDB) using PyMol. Surface representation of the three PDB structures used for docking analysis. Figure made using PyMol. (1) MR8 chain A (yellow) and chain B (orange); drug-binding cavity in cyan; (2) 1IRJ chain A (purple) and chain B (pink); yellow is the original Figure 1. The molecular structure of the four drugs, aspirin, celecoxib, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CPS) dexamethasone and diclofenac, used in the docking study. binding site, but when CPS is removed during docking, all the selected drugs bind there; cyan depicts the binding site of aspirin and diclofenac only (in the presence of CPS in the opposite cavity); (3) 4GGF S100A8: chain A and K (red), S100A9 chain C and L (dark pink), drug-binding Affinity (grid) maps of 20×20×20 Å grid points and 0.375 Å binding cavity in cyan. site grid generation spacing were generated using the Autogrid program (43). AutoDock parameter set- and distance-dependent dielectric functions were used in the calculation of the van der Waals’ and the electrostatic terms, respectively. Docking simulations were performed using the Lamarckian genetic algorithm (LGA) and the Solis and Wets local search method (44). Initial positions, orientation, and torsions of the ligand molecules were set randomly. Each docking experiment was derived from 10 different runs that were set to terminate after a maximum of 250000 energy evaluations. The population size was set to 150. During the search, a translational step of 0.2 Å, and quaternion and torsion steps of 5 were applied in the current series of docking analysis.

Results The main focus of the present study was to discover novel anticancer drug targets by transcriptomic profiling and to identify possible protein−drug interactions by molecular docking analysis. We identified S100A8 as an important protein in kidney cancer and attempted to demonstrate its potential as an anticancer drug target. Identification of differentially expressed genes. We profiled fresh kidney tissue specimens and compared them with normal control samples. We performed PCA scatter plot analysis for visualizing the high-dimensional array data where each point represents a Figure 3. Hierarchical clustering and functional analysis of genes chip or sample. The results of PCA of transcriptomic data significantly differentially expressed in kidney cancer using Affymetrix showed that the samples from the same tissue type clustered Human ST 1.0 array and Partek GS 6.6 software. tightly together. Clear differences were also observed between tumors and normal tissues, revealing distinct expression profiles for each tissue type. Comparison of the genome-wide expression of kidney cancer revealed 1,335 differentially expressed genes: Pathways and networks underlying kidney cancer. To understand 852 up-regulated and 483 down-regulated, with a 2-fold or the mechanisms by which the genes alter a wide range of greater change and FDA of p<0.05 (Figure 3, Table II). physiological processes, we examined biological functions,

1876 Mirza et al: S100A8: An Anticancer Target for Kidney Cancer ation in the LXR/RXR pathway. Red color represents ation in the LXR/RXR pathway. Liver X receptor/retinoid X receptor(LXR/RXR) activation: Transcriptomic signatures of kidney cancer showed a significant activ a significant cancer showed of kidney signatures Transcriptomic activation: X receptor(LXR/RXR) X receptor/retinoid Liver overexpression and green color underexpression. and green overexpression Figure 4.

1877 ANTICANCER RESEARCH 34: 1873-1884 (2014)

Figure 5. 2D plot of inhibitors of S100A8, S100A9 and S100A8−S100A9 protein interaction profiles by DockingServer. Ligand bonds, non-ligand bonds, hydrogen bonds and their lengths are marked for aspirin, celecoxib, dexamethasone and diclofenac. A-D denote interaction of aspirin, E-H of celecoxib, I-L of dexamethasone and M-P of diclofenac with S100A8 (1MR8), S100A9 (1IRJ, 1IRJ-CPS), and S100A8−S100A9 (4GGF), respectively. molecular network and pathways associated with kidney cancer. receptor/retinoid X receptor activation, Interleukin-12 signaling Interestingly, cellular movement was significantly over- and production in macrophages, production of nitric oxide and represented as a process for both down-regulated and up- reactive oxygen species in macrophages, notch signaling, and regulated genes, indicating that metastasis is probably linked to clathrin-mediated endocytosis signaling (Figure 4, Table III). a different equilibrium of switching on and off i.e. cell division Extensive pathway analysis of differentially regulated genes may cascade is on while tumor suppressor cascade is off. Functional provide novel hypotheses underlying tumor invasion and analysis of kidney cancer-associated genes showed an metastatic progression of kidney cancer. overexpression of genes involved in cell-cycle progression, DNA repair, cell death, tumor morphology and tissue development. Docking studies. Molecular docking studies predicted potential Pathway analysis showed significant disruption in certain interactions of our proposed protein drug target with the selected signaling pathways, including atherosclerosis signaling, liver X drug molecules. As far as we are aware of, this is the first

1878 Mirza et al: S100A8: An Anticancer Target for Kidney Cancer

Figure 6. Interactions of ligand with the protein. Red represents protein as cartoon; grey represents interacting side chain as cylinder; and green represents drug as ball and stick model.

Table II. Significantly differentially expressed genes in kidney cancer. Negative fold change value indicate down-regulation of gene.

Gene Symbol Gene Name p-Value Fold-change

CXCL10 Chemokine (C-X-C motif) ligand 10 0.0075294 13.1003 LYZ Lysozyme 0.0291376 12.3279 ANO4 Anoctamin 4 0.0324189 11.9335 TIMD4 T-cell immunoglobulin and mucin domain containing 4 0.0016176 11.3754 NETO2 Neuropilin (NRP) and tolloid (TLL)-like 2 0.0017682 10.6009 PTGS1 Prostaglandin-endoperoxide synthase 1 0.0003123 9.71631 CAV1 Caveolin 1, caveolae protein, 22kDa 0.0128379 9.15235 EBF2 Early B-cell factor 2 0.0006310 8.39571 PLA2G7 Phospholipase A2, group VII (platelet-activating factor acety 0.0004207 8.03755 AHNAK2 AHNAK nucleoprotein 2 0.0025453 7.73542 RGS1 Regulator of G-protein signaling 1 0.0274691 7.69189 IGF1 -like growth factor 1 (somatomedin C) 0.0281717 6.89465 MYBL1 v-myb Myeloblastosis viral oncogene homolog (avian)-like 1 // 0.0066664 6.8015 VCAN Versican 0.0350255 6.75548 TOPBP1 Topoisomerase (DNA) II binding protein 1 1.11E-06 2.05612 TDO2 Tryptophan 2,3-dioxygenase 7.91E-06 3.30054 FOXM1 Forkhead box M1 8.08E-06 2.89359 S100A8 S100 calcium binding protein A8 0.0159132 2.66364 ANKRD13A Ankyrin repeat domain 13A 3.83E-05 2.78943 CALB1 Calbindin 1, 5.79E-05 –158.598 UMOD Uromodulin 1.66E-05 –150.524 PLG Plasminogen 0.00027811 –74.3902 ALDOB Aldolase B, fructose-bisphosphate 0.00068977 –65.1105 SLC12A3 Solute carrier family 12 (sodium/chloride transporters), member 8.17E-05 –61.4283 KNG1 Kininogen 1 0.00316824 –56.3974 NPHS2 Nephrosis 2, idiopathic, steroid-resistant (podocin) 1.56E-05 –41.0848 AQP2 Aquaporin 2 (collecting duct) 0.00039755 –27.0564

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Table III. Canonical pathways predicted by Ingenuity Pathway Analysis for genes significantly differentially expressed in kidney cancer.

Ingenuity Canonical Pathways -log(p-value) Molecules

Atherosclerosis signaling 3.14E00 APOE, APOM, MSR1, PLA2R1, PLA2G7, SELPLG, COL1A2, APOC1, APOL1, COL1A1, IL18, ALB, LYZ, CCL2, S100A8, PDGFD, RBP4, COL3A1 LXR/RXR Activation 2.93E00 KNG1, SCD, APOE, APOM, ECHS1, MSR1, AMBP, ABCG1, APOC1, APOL1, IL18, ALB, LYZ, LY96, CCL2, S100A8, HADH, RBP4 IL-12 Signaling and 1.88E00 PPARG, APOE, APOM, PIK3C2A, MST1, APOL1, PIK3R3, APOC1, ALB, LYZ, production in macrophages IL18, TGFB2, MAP3K8, S100A8, PRKCH, RBP4 Production of nitric oxide and 1.04E00 APOE, APOM, PIK3C2A, JAK2, APOC1, APOL1, PIK3R3, NCF1, ALB, reactive oxygen species in macrophages LYZ, NCF2, PPP1R12A, MAP3K8, S100A8, PRKCH, FNBP1, RBP4 Clathrin-mediated endocytosis signaling 8.38E-01 APOE, APOM, PIK3C2A, EGF, FGF1, APOL1, PIK3R3, APOC1, ALB, LYZ, ARRB2, CBL, IGF1, S100A8, PDGFD, RBP4

The table shows the significantly overrepresented canonical pathways across the whole dataset of differentially expressed genes.

structural attempt to study possible binding for cancer and also by Val 80 (B). Interestingly, the three fluorine residues therapeutics. To understand the molecular interaction between (H-bond acceptors) of celecoxib orient and form halogen bonds drugs and S100A8, a series of molecular docking analyses were with Gln 69 (A). performed using three-dimensional structures available (PDBID: The binding ability of dexamethasone is limited as there are 1IRJ, 1MR8, and 1XK4) with four known anti-inflammatory no H-bonds formed and the free energy of the bound structure is drugs namely aspirin, celecoxib, dexamethasone and diclofenac. −2.02 kcal/mol. However, there are around 17 hydrophobic Molecular docking revealed that all four drugs are able to bind in interactions with the aliphatic non-polar hydrophobic residues of the ligand-binding domain of these S100 proteins. The ligand- chain A Ile 73, Ile 76 and Val 80. Other contacts are mediated binding site was a hinge region containing two EF-hand motifs. by polar residues Lys 77 (A) and Gln 69 (B) and the remaining Based on their size, stereochemistry and structural differences, hydrophobic amino acids. the ligands exhibited different intensities in binding with the Diclofenac shows good binding characteristics with the protein target molecules. The predicted parameters of estimated S100A8 dimer but forms neither H-bonds nor polar contacts. binding free energy, inhibition constant (Ki), total energy of van Estimated free energy of binding is predicted to be −4.32 der Waals’, hydrogen bonding, desolvation, electrostatic energy, kcal/mol. It mainly shows hydrophobic interactions: two with total intermolecular energy and interacting surface area were Leu 72 (B), one with Ile 73 (A), 12 with Ile 76 (A) and three evaluated to estimate the favorable binding of ligand drug with Val 80 (A). The remaining protein residues involved in other molecules to the target protein. Molecular visualization was contacts are Ile 76 (A), Lys 77 (A) and Val 80 (A). All of these performed using PyMol. Complete interaction profiles (hydrogen residues are at a maximum distance 3.1-3.8Å. bonds, polar, hydrophobic, pi−pi, cation−pi and other contacts), and hydrogen bonding interactions (HB plot) were also studied (S100A9)2: 1IRJ (chain A and B). In the docked structure there (Figure 5 and 6, Table IV). were no H-bonds but there were two hydrophobic interactions with Ile 16 (B) amongst other attractive contacts. We had not (S100A8)2: 1MR8 (chain A and B). Aspirin binds at the ligand removed the present CHAPS (CPS) molecule from the crystal binding site without forming any H-bonds but displayed two structure taken. Aspirin owing to its small shape and size was polar contacts with the residue Gln 69 (A) and several able to fit into the binding groove. Interestingly, aspirin binds to hydrophobic and van der Waals’ interactions mainly with the the S100A9 dimer at a totally new site different from the cavity hydrophobic residues Leu 72 (A), Ile 73 (A), Ile 76 (B) and also where CPS is present. The drug-binding cavity was lined mainly with Gln 69 (A). Predicted free energy of binding is −3.26 by hydrophobic residues: Ile 16, His 20 (B), Phe 76, Ala 84 (A) kcal/mol. and Leu 86. Glu 77 also exhibited indirect interactions. Attractive Celecoxib binds to this protein molecule in a much better way stacking pi−pi interactions between aromatic protein residues compared to aspirin, with Ki of 960.05 μM. It is able to form H- present, i.e. His 20 and Phe 76, were also noted. bonds with a critical amino acid residue in the ligand binding Docking study in the presence of CPS and the docked domain site of S100A8 i.e. Gln 69 (A), which also forms three structure has a positive free energy of binding, implying that more polar contacts with the chosen ligand. More than two binding is not feasible as most of the decomposed interaction dozen hydrophobic and other interactions are also predicted to energies are positive. Enhanced docking properties can possibly be mediated by Gln 69 (A), Leu 72 (A), Ile 73 (A), Ile 76 (B) be achieved by increasing the simulation box size and making

1880 Mirza et al: S100A8: An Anticancer Target for Kidney Cancer

Table IV. Docking results (binding and interaction values) for docking of S100A8, S100A9, S100A8−S100A9 with aspirin, celecoxib, dexamethasone and diclofenac.

Aspirin Celecoxib Dexamethasone Diclofenac

(S100A8)2 (1MR8) Est. free energy of binding (kcal/mol) −3.26 −4.12 −2.02 −4.32 Est. Inhibition Constant, Ki (mM) 4.11 0.960 33.01 0.680 vdW + Hbond + desolv energy (kcal/mol) −4.27 −5.94 −3.95 −5.58 Electrostatic energy (kcal/mol) −0.05 −0.02 +0.07 −0.05 Total intermol. energy (kcal/mol) −4.31 −5.96 −3.88 −5.62 Frequency 10% 10% 20% 30% Interact. surface (square angstrom) 399.423 608.2 553.878 491.597

(S100A9)2 (1IRJ) Est. free energy of binding (kcal/mol) −3.26 +96.99 +2.63 −3.24 Est. Inhibition Constant, Ki (mM) 4.09 −−−−− −−−−− 4.22 vdW + Hbond + desolv energy (kcal/mol) −4.37 +95.40 +1.15 −4.38 Electrostatic energy (kcal/mol) −0.02 −0.09 −0.26 −0.06 Total intermol. energy (kcal/mol) −4.40 + 95.31 +0.89 −4.44 Frequency 70% 20% 10% 60% Interact. surface (square angstrom) 396.622 633.081 614.496 542.149

(S100A9)2 (1IRJ-CPS) Est. free energy of binding (kcal/mol) −3.20 −4.97 −5.51 −5.01 Est. Inhibition Constant, Ki (mM) 4.49 0.227 0.091 0.214 vdW + Hbond + desolv energy (kcal/mol) −4.04 −6.40 −6.30 −5.61 Electrostatic energy (kcal/mol) −0.01 −0.05 −0.10 +0.00 Total intermol. energy (kcal/mol) −4.05 −6.46 −6.40 −5.61 Frequency 30% 10% 20% 10% Interact. surface (square angstrom) 396.227 564.476 487.061 494.413

(S100A8−S100A9)2 (4GGF) Est. free energy of binding (kcal/mol) −4.40 −3.01 −4.92 −4.95 Est. Ki (mM) 0.597 6.25 0.245 0.234 vdW + Hbond + desolv energy (kcal/mol) −4.96 −4.54 −5.23 −5.52 Electrostatic energy (kcal/mol) −0.22 −0.01 −0.05 −0.01 Total intermol. energy (kcal/mol) −5.18 −4.55 −5.27 −5.53 Frequency 90% 80% 60% 40% Interact. surface (square angstrom) 496.03 756.175 486.055 455.318

the ligand-binding domain free of any pre-existing bound ligand Aspirin binds with a low estimated free energy of binding of such as CPS in this case. −3.20 kcal/mol with chain B and there is no H-bond formation Similarly to the above case, docking was carried out in the between the drug and target protein molecule. The drug-binding presence of CPS and the docked structure has a positive value site seems to be different from that of the above case in the for free energy of binding, indicating that binding is not presence of CPS and aspirin binds at the binding site previously feasible. Better docking results are expected perhaps by occupied by CPS. Five hydrophobic interactions can be seen with increasing the simulation box size and making the ligand- Ile 62 and one with Leu 82. In addition, around 12 other contacts binding domain lined by amino acids Thr 68, Glu 77, Met 81 are observed with Asp 65, His 61, Glu 52, Arg 85 and Val 58. and Arg 85 free of any pre-existing bound ligand. Hence, Ki Celecoxib binds at the CPS-binding site of (S100A9)2 as cannot be estimated in this case. shown in the figure made by using PyMol (Figure 6). There The binding of diclofenac molecule was stabilized with the are two H-bonds between Phe 48 and Arg 85 belonging to help of three H-bonds, one with the residue Thr 68 (B) and two chain B of the protein molecule at a distance of 3.35Å and with Arg 85 (B), one polar contact with Thr 68 (B), one halogen 2.57Å. In addition, polar contacts (Arg 85), hydrophobic bond, and few hydrophobic interactions (van der Waals’ contacts) interactions (Leu 49 and Leu 86), van der Waals’ and other primarily with Met 81 (B) (distances upto 3.7Å) and Glu 77 (A). interactions (Phe 48, Lys 51, Glu 52 and Leu 86) are noted. It seems that diclofenac would bind at a site adjacent to the Our results clearly demonstrate the binding ability of aspirin-binding cavity (not shown). dexamethasone with critical amino acid residues of the ligand-

1881 ANTICANCER RESEARCH 34: 1873-1884 (2014) binding domain of chain B only, potentiating inhibitory ability. Discussion The interacting residues of the drug-binding site are Leu 49, Glu 52, Val 58, His 61, Ile 62 and Leu 86. However, there are no H- Kidney cancer includes heterogeneous tumors with diverse bonds. Stacking attractive pi−pi interactions with His 61 and two molecular and clinical characteristics that is reflected in their carbon atoms of the drug are also found. Several polar, response to specific treatments. In the present work, we hydrophobic, van der Waals’ and other contacts are seen in the identified S100A8 as a potential biomarker for kidney cancer and docked complex. The estimated free energy of binding is in silico analysis shows that aspirin, celecoxib, dexamethasone predicted to be −5.51kcal/mol. and diclofenac are predicted to bind to S100A8, presumably The diclofenac molecule is predicted to have good affinity and inhibiting downstream signaling in kidney cancer. Our finding binding with the S100A9 dimer but without any H-bond leads to the hypothesis that S100A8 is a promosing anticancer formation. Around 15 hydrophobic interactions are seen with the drug target and aspirin, celecoxib, dexamethasone and diclofenac non-polar residues lining the binding groove, i.e. Leu 49, Val 58, are S100A8 inhibitors. Ile 62, Leu 82 and Leu 86. Stacking pi−pi interactions with His S100 proteins participate in numerous functions including 61 and the C14 atom of the drug are present. Asp 65 forms a protein phosphorylation, enzymatic activation, calcium halogen bond with Cl1 of diclofenac, with the distance between homeostasis, and interaction with cytoskeletal components (45). them being 3.49Å. Remaining weak interactions are found for Most genes encoding S100 proteins are clustered on a region of Glu 52, Val 58, His 61, Asp 65 and Arg 85. The estimated human 1q21 that is prone to chromosomal re- inhibition constant Ki is 214.31 μM. arrangements, suggesting a link of S100A8 and S100A9 proteins with metastasis and tumor formation (45-46). Abnormal (S100A8−S100A9)2 (4GGF). Aspirin interacts only with the C expression of S100 proteins, including S100A8 and S100A9, and L chains of S100A9, exhibiting only polar contacts and were observed in a variety of different cancer types, such as hydrophobic interactions and no direct H-bonds. In total, three gastric, lung, breast, liver, pancreatic and squamous esophageal polar contacts are predicted, one with Glu 92 (C) and two with carcinomas (9-11, 14-16, 47-50). Despite elevated expression Arg 85 (L). Leu 82 and 86 create hydrophobic contacts and are and the distinct role of S100A8 in different cancer types, less is at a distance of 3.81Å and 3.89Å, respectively. known about the expression status or role of S100A8 in the Docking results for celecoxib are promising, with estimated progression of kidney cancer. free energy of binding of −3.01 kcal/mol, and pairwise S100A8 has cell growth-promoting activity at low decomposition of energies of interacting residues are zero. concentrations by binding to RAGE. In addition, RAGE binding Similar to the case of aspirin binding, the ligand celecoxib is to S100A8−S100A9 promotes phosphorylation of LIM (cell found in the binding site made by chains C and L of S100A9 and lineage protein 11 (Lin-11), islet-1 (ISL1), Mechanosensory interacts with different residues present. It forms a total of six H- protein 3 (MEC-3)), domain kinase. This phosphorylation is a bonds: three with Glu 52 (L), two with Glu 92 (C) and one with critical step in cofilin recycling and actin polymerization. Gly 97 (C). Several hydrophobic interactions are seen with the Interestingly, RAGE binding to S100A8−S100A9 enhanced cell hydrophobic amino acids of chain L: Leu 49, Ile 62, Leu 82, and mesenchymal properties and induced epithelial−mesenchymal Leu 86. Six halogen bonds with fluorine residue of the ligand transition. Moreover, RAGE binding to S100A8−S1009 played were seen, most of which were water-mediated. More than 12 an important role in promoting invasion and metastasis in cancer other contacts were noted, formed mainly with the polar residues (9, 16). These studies indicate the potential of S100A8 as an such as Arg 85 (L), Trp 88 (C), His 91 (C) and Glu 96 (C). anticancer target. Dexamethasone binds very well at the site lined mainly by In our docking study of the S100A8 dimer with the four chain L (S100A9) residues and partly by chain K (S100A8). The selected drugs, the best overall binding was exhibited in terms docked structure does not show any H-bonding nor polar of estimated free energy of binding and Ki value by diclofenac contacts. More than 12 hydrophobic interactions are possible, followed by celecoxib and aspirin, and the least by mediated by amino acids His 20 (L), Val 24 (L), Pro 29 (L) and dexamethasone. Binding of aspirin with S100A9 dimer does His 87 (K). Other noticeable interactions are with Gln 21 and not show much difference in terms of energy and Ki in the Ser 23. presence or absence of CPS. S100A9 dimer also seems to Diclofenac binds best with the selected chains of the have multiple ligand-binding areas as exhibited by the calprotectin complex structure taken for docking analysis and all different ligand-binding sites for aspirin and diclofenac in the the interacting residues belong to chain L (S100A9). It does not presence, as well as absence, of already bound ligand, CPS. form H-bonds but Val 24 and Pro 29 are predicted to have good However the CPS-binding cavity is quite hydrophobic and is hydrophobic interactions, with a maximum distance of 3.7 Å. the most preferable binding site for compounds having Interestingly, His 20 displays six stacking ring interactions and compatible shape and stereochemistry. Aspirin does not form also one halogen bond. Two other contacts are predicted with H-bonds in any of the docked structures but it displayed the Asn 17, His 20, Gln 21, Ser 23 and Val 24. best binding capability with calprotectin. The binding results

1882 Mirza et al: S100A8: An Anticancer Target for Kidney Cancer for dexamethasone and diclofenac are similar with calprotectin BIO1258-03 and 10-BIO1073-03). The Authors would like to thank complex and neither of them forms H-bonds with the protein Nuha Alansari, Alaa Albogmi, Manal Shaabad, Amal M. Noor and target. Even though celecoxib is able to form six H-bonds with Manar Ata for sample collection, performing RNA extraction, bioanalyzer assays and microarray experiments. We thank the S100A8−S100A9 heterotetramer, its estimated free energy of patients, physicians, nurses, and pathologists of the King Abdulaziz binding is comparatively low. University Hospital and King Faisal Specialist Hospital and Research This was a pioneering structure-based approach to study Center, Jeddah, Saudi Arabia. S100A8 protein interactions with the chosen anti- inflammatory drugs at the molecular level. The References computational results provide valuable insights into the binding modes of the four tested inhibitors to the 1 Hanahan D and Weinberg RA: Hallmarks of cancer: The next generation. S100A8−S100A9 complex and the key factors affecting Cell 144(5): 646-674, 2011. binding affinity. It has been demonstrated that the 2 Hanahan D and Weinberg RA: The hallmarks of cancer. Cell 100: 57- hydrophobic interactions and hydrogen bonding with 70, 2000. 3 Donato R, Cannon BR, Sorci G, Riuzzi F, Hsu K, Weber DJ and Geczy S100A8 make pivotal contributions to the binding structures CL: Functions of S100 proteins. Curr Mol Med 13: 24-57, 2013. and binding free energies, although the van der Waals’ and 4 Donato R: S100: A multigenic family of calcium-modulated proteins of electrostatic interactions also significantly contribute to the the EF-hand type with intracellular and extracellular functional roles. Int stabilization of the binding structures. The calculated binding J Biochem Cell Biol 33: 637-668, 2001. free energies are in good agreement with the available 5 Marenholz I, Heizmann CW and Fritz G: S100 proteins in mouse and man: from evolution to function and pathology (including an update of the experiment activity data. The detailed structural insight, nomenclature). Biochem Biophys Res Commun 322: 1111-1122, 2004. binding modes and the crucial factors affecting the binding 6 Hessian PA, Edgeworth J and Hogg N: MRP-8 and MRP-14, two free energies obtained from the present computational studies abundant Ca(2+)-binding proteins of neutrophils and monocytes. J may provide valuable insights for future rational structure- Leukoc Biol 53: 197-204, 1993. based design of novel, more potent S100A8 inhibitors. 7 Kerkhoff C, Voss A, Scholzen TE, Averill MM, Zänker KS and Bornfeldt KE: Novel insights into the role of S100A8/A9 in skin biology. Exp Dermatol 21: 822-826, 2012. Conclusion 8 Passey RJ, Williams E, Lichanska AM, Wells C, Hu S, Geczy CL, Little MH and Hume DA. A null mutation in the inflammation-associated S100 Our analysis suggests distinct transcriptomic signatures for protein S100A8 causes early resorption of the mouse embryo. J Immunol kidney cancer, with significantly high levels of S100A8 163: 2209-2216, 1999. expression pointing to one of the underlying molecular 9 Yin C, Li H, Zhang B, Liu Y, Lu G, Lu S, Sun L, Qi Y, Li X and Chen W: RAGE-binding S100A8/A9 promotes the migration and invasion of mechanisms contributing to progression of kidney cancer. human breast cancer cells through actin polymerization and Although further validation is needed to corroborate these epithelial−mesenchymal transition. Breast Cancer Res Treat 142(2): 297- findings, analysis of kidney cancer tissue is a promising tool 309, 2013. for identifying biomarkers of interest. Protein−ligand 10 Turovskaya O, Foell D, Sinha P, Vogl T, Newlin R, Nayak J, Nguyen M, interaction studies play a vital role in structure-based Olsson A, Nawroth PP, Bierhaus A, Varki N, Kronenberg M, Freeze HH and Srikrishna G: RAGE, carboxylated glycans and S100A8/A9 play computational drug design. Our docking-based findings shed essential roles in colitis-associated . Carcinogenesis 29: insight into S100A8 protein as a potential target for 2035-2043, 2008. therapeutic intervention in kidney cancer. S100A8 has been 11 Grebhardt S, Müller-Decker K, Bestvater F, Hershfinkel M and Mayer identified as an attractive target for anticancer drug D: Impact of S100A8/A9 expression on prostate cancer progression in development due to its central role in mediating inflammatory vitro and in vivo. J Cell Physiol 229(5): 661-671, 2014. 12 Hermani A, De Servi B, Medunjanin S, Tessier PA and Mayer D: pathways. Further investigations such as quantitative S100A8 and S100A9 activate MAP kinase and NF-κB signaling structure−activity relationship studies are required to pathways and trigger translocation of RAGE in human prostate cancer determine more favorable interaction with S100A8 and its cells. Exp Cell Res 312: 184-197, 2006. partners. Better binding ligands with more affinity and efficacy 13 Rafii S and Lyden D. S100 chemokines mediate bookmarking of pre- can be further designed and validated using combinatorial metastatic niches. Nat Cell Biol 8: 1321-1323, 2006. chemistry and co-crystallization approaches. 14 Yong HY and Moon A: Roles of calcium-binding proteins, S100A8 and S100A9, in invasive phenotype of human gastric cancer cells. Arch Pharm Res 30: 75-81, 2007. Conflicts of Interest 15 Németh J, Stein I, Haag D, Riehl A, Longerich T, Horwitz E, Breuhahn K, Gebhardt C, Schirmacher P, Hahn M, Ben-Neriah Y, Pikarsky E, The Authors declare that there are no conflicts of interest. Angel P and Hess J: S100A8 and S100A9 are novel nuclear factor kappa B target genes during malignant progression of murine and human liver Acknowledgements carcinogenesis. Hepatology 50: 1251-1262, 2009. 16 Chen KT, Kim PD, Jones KA, Devarajan K, Patel BB, Hoffman JP, Ehya This work was supported by King Abdullah City for Science and H, Huang M, Watson JC, Tokar JL and Yeung AT: Potential prognostic Technology, Riyadh, Saudi Arabia (KACST, Strategic Project ID. 10- biomarkers of pancreatic cancer. Pancreas 43(1): 22-27, 2014.

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K, Ichida F, Foell D, Kehrel B, Gerke V, Sorg C and Roth J: Myeloid- 50 El-Rifai W, Moskaluk CA, Abdrabbo MK, Harper J, Yoshida C, Riggins related proteins 8 and 14 induce a specific inflammatory response in GJ, Frierson HF Jr. and Powell SM: Gastric cancers overexpress S100A human microvascular endothelial cells. Blood 105: 2955-2962, 2005. calcium-binding proteins. Cancer Res 62: 6823-6826, 2002. 31 Sunahori K, Yamamura M, Yamana J, Takasugi K, Kawashima M, Yamamoto H, Chazin WJ, Nakatani Y, Yui S and Makino H: The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 Received January 14, 2014 mitogen-activated protein kinase in rheumatoid arthritis. Arthritis Res Revised February 17, 2014 Ther 8: R69, 2006. Accepted February 18, 2014

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