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Review Article Head and Neck

Proteomic Advances and Achievements

Taia Maria Berto Rezende, PhD; Mirna de Souza Freire, BS; and Octa´vio Luiz Franco, PhD

Tumors of the head and neck comprise an important neoplasia group, the incidence of which is increasing in many parts of the world. Recent advances in diagnostic and therapeutic techniques for these lesions have yielded novel molecular targets, uncovered signal pathway dominance, and advanced early cancer detection. Proteomics is a powerful tool for investigating the distribution of proteins and small molecules within biological systems through the analysis of different types of samples. The proteomic profiles of different types of cancer have been studied, and this has provided remarkable advances in cancer understanding. This review covers recent advances for ; it encompasses the risk factors, pathogenesis, proteomic tools that can help in understanding cancer, and new proteomic findings in this type of cancer. Cancer 2010;116:4914–25. VC 2010 American Cancer Society.

KEYWORDS: head and neck cancer, proteomics, cancer risk factors, cancer pathogenesis, diagnostic biomarkers.

Head and neck cancer (HNC) is a term that encompasses epithelial malignancies that arise in the , , oral cavity, , and . Almost all of these epithelial malignancies are squamous cell (SCCs) of the head and neck (HNSCCs), for which the most important risk factors are and consump- tion.1 HNSCCs initiate from the mucosal surface, whereas non-SCCs initiate from other cell types (eg, that come from glandular cells). Successful treatment of those patients depends on early detection and correct therapy for each cancer phase. Development of novel, potentially efficacious anticancer agents and identification of biomarkers depend on further elucidation of molecular events in HNC development.2 Cancer can be regarded as a deviation from normal signaling toward a more deregulated state that results in cellular proliferation.3 Although each individual mutation may induce specific cancer phenotypes, it is the interaction of the functional changes in transcription and proteins that produce the characteristics of cancer.4 Alteration in the content and functional state of the proteins with many linkages may shift the equilibrium state of the protein-signaling network to enhance a survival advantage of the affected cells. Searching for such hub proteins is the main purpose of cancer proteo- mics. Low-abundance tissue-specific proteins can be useful as diagnostic markers.3 The focus of this review is to discuss general aspects of HNC in the light of proteomics, a potent biological tool that can help in early cancer diagnosis, treatment, and prognosis.

Epidemiology The number of new cases of HNC in the United States was 40,490 in 2006, accounting for about 3% of adult malignan- cies. The worldwide incidence exceeds half a million cases annually.5 Oral SCC (OSCC) is the most common HNC and accounts for >300,000 new cancer cases worldwide annually.6 Cancer of the upper aerodigestive tract ranks as the third most frequent group of among males and the fourth most frequent among females in developing countries. High-risk regions for oral cavity cancer include Melanesia (subregion of Oceania) and south-central Asia, western and southern Europe, and southern Africa. For , southern and eastern Europe, South America, and western Asia stand out.7 A slight decrease in the overall incidence of HNC has been detected in the past 2 decades; however, an increase in cancer in the base of the and tonsillar cancer has been noted, which could be more pronounced in young adults in the USA and European countries.8

Corresponding author: Octa´vio Luiz Franco, SGAN Quadra 916, Mo´dulo B, Av. W5 Norte. 70.790-160-Brası´lia – DF, Brazil; Fax: (011) 55-61-3347-4797; [email protected] Center of Proteomic and Biochemical Analyses, Post-Graduation in Biotechnology and Genomical Sciences, Catholic University of Brasilia, Brasilia-DF, Brazil DOI: 10.1002/cncr.25245, Received: August 27, 2009; Revised: November 10, 2009; Accepted: January 5, 2010, Published online July 21, 2010 in Wiley Online Library (wileyonlinelibrary.com)

4914 Cancer November 1, 2010 Head and Neck Cancer and Biomarkers/Rezende et al

Main HNC Risk Factors ence of low levels of carcinogenic substances in alcoholic The risk factors for HNC are especially represented by beverages; or 3) cellular injury produced by ethanol tobacco consumption in various forms (smoked or metabolites.17 Alcohol acts independently, as well as in chewed) and use of the quid—a preparation of synergy with tobacco, in oral . Tobacco and various ingredients, including tobacco and the seeds of the alcohol effects are not only risk factors for developing betel palm, wrapped in a betel leaf. The upper aerodiges- primary cancer, but also for developing second primary tive tract is the first compartment of the human body that .18 Therefore, avoiding tobacco and alcohol is the makes contact with the harmful components of tobacco most desirable way not only to prevent primary oral smoke. The passage of the smoke, whether in an active or cancers, but also to reduce risk of second cancers of the passive smoker, determines some alterations in the aero- aerodigestive system. digestive area that is directly exposed to its harmful action. HNSCC has also been associated with genetic poly- Tobacco contains more than 50 components, including morphisms in genes that encode human enzymes related aromatic polycyclic hydrocarbon, tobacco specific nitro- to toxic substance metabolism, and that also affect the samines, aromatic amines, and aldehydes, which are individual’s susceptibility to noxious effects of carcino- responsible for neoplastic buccal transforma- genic chemistry.19 Bondy et al demonstrated in vitro that tion.9 One of the first steps in the development of head mutagenicity assays suggest that familial factors may be and neck neoplasia may be the link between these com- important in a predisposition to HNSCC.20 Conversely, pounds and the mucosal cell’s DNA.10 The metabolism human papillomavirus has been reported to be associated of chemicals occurs in 2 phases. In phase 1, hydrolyzes, with HNSCC in patients without a or reductions, and oxidization reactions occur mainly by alcohol consumption.21 the enzymatic group P-450, producing reactive and Diet and nutrition are also risk factors for HNSCC. toxic substances. The induces glutathione Although the individual responsible micronutrients have S-transferase transcription to eliminate the xenobiotics.11 not been formally identified, vegetables and fruits Although most of these enzymes are hepatic, there is that protect against and precancer are rich in evidence of them in the buccal mucosal, where they de- b-carotene, C, and , with antioxidant velop their oxidative activity.12 The toxic metabolites pro- properties.22 Epidemiological studies from northern duced in phase 1 link to macromolecules such as DNA, China, parts of Africa, and Iran show environmental RNA, and protein-forming complexes that promote factors including nutritional zinc deficiency, which is genetic instability, mutation, and the beginning of the associated with a restricted diet and exposure to carcino- carcinogenesis process.13 Phase 1 products may be directly genic N-nitrosamines, in the etiology of .23 excreted or undergo phase 2 enzyme action. Glutathione S-transferases are the main enzymatic components Pathogenesis responsible for the metabolism of phase 2. After the Molecular changes in HNC occur through multiple steps, glucuronidation, sulfation, methylation, and conjunction each characterized by the sequential stimulation of addi- reactions, the xenobiotic agents are inactivated and tional genetic defects, followed by clonal expansion. The become hydrosoluble, being easily excreted.13 genetic alterations observed are mainly because of onco- Some studies link cytochrome P450 family 1, sub- gene activation and inactivation family A (CYP1A1) and CYP2E1 genotype to a suscepti- leading to deregulation of cell proliferation and death.24 bility to oral cancer, but others have failed to confirm this The histological evolution from normal epithelium to association.14 Jaber et al observed that tobacco has an in- invasive includes , mild , dependent role in the etiology of oral epithelial dysplasia, and severe dysplasia or in situ carcinoma.2 although alcohol is principally only important in conjunc- Growth regulators and tumor suppressor genes act tion with tobacco use.15 However, Schlecht et al observed as transducers of negative growth signals. Genetic altera- among nonsmokers that alcohol in fact acts both as tions involving the tumor suppressor genes p16 and an independent and as a promoter of oral, are frequently observed in HNC.24 Inactivating the p16 pharyngeal, and esophageal cancers.16 Alcohol is a cancer gene might confer cell growth defects, contributing to the promoter via 1 or more of the following mechanisms: 1) tumorigenic process, because of its involvement in cell increased permeability of mucosal cells to cycle regulation, including cell cycle arrest and apoptosis. because of solubilization by alcohol; 2) pres- Conversely, TSG p53 has a role in maintaining genomic

Cancer November 1, 2010 4915 Review Article stability, cell cycle progression, cellular differentiation, be responsible for their active spread.35 As a consequence DNA repair, and apoptosis, so it is called the ‘‘guardian of of Th2-like cytokines’ profile, a suppression of Th1 cyto- the genome.’’25 Moreover, p53 plays an important role in kine genes was reported to increase tumor load and lymph cell-cycle control and in the induction of apoptosis. The node .24 Bose et al36 showed low levels of Th1 gene can be inactivated by several mechanisms, including cytokine (IL-12 and interferon [IFN]c), and high levels of occasional mutations, deletions, and binding with cellular Th2 cytokine (IL-4 and IL-10) secretion in culture super- and viral proteins in different kinds of cancer, among natant of HNSCC peripheral blood mononuclear cells. them SCC.25 According to this study, the combination of Th2 cyto- Moreover, epidermal growth factor receptor kines is able to suppress temporarily the generation of (EGFR) protein expression is elevated in 90% or more IFNc-producing cells without affecting the polarity of T cases of HNSCC,26 developing a central role in HNSCC cells, and these actions occurred via STAT1 and STAT4 biology.27 The EGFR ligand binding results in a molecu- activation.36 Other important cytokines involved in lar cascade, covering the receptor-linked tyrosine kinase HNSCC include IL-27, which has a potent ability to activation and many downstream pathways, which induce antitumor-specific activity and is mainly mediated þ regulate proliferation, differentiation, migration/motility, through CD8 T cells with enhanced cytotoxic T adhesion, prevention of apoptosis, angiogenesis, meta- lymphocytes activity,37 and granulocyte macrophage static potential, enhanced survival, and gene transcrip- colony-stimulating factor, which presents antitumor tion.2 To date, 3 major mitogen-activated protein kinase activity.38 As shown, the cytokines involved in HNSCC (MAPK) subfamilies have been described: extracellular play an important role in this process and could be signal-regulated kinase (ERK) 1 and 2, p38-MAPK, and targeted for . c-jun amino-terminal kinase stress activated protein kinase, although other MAPK members have been identi- fied (eg, ERK5 and ERK7).28 Each MAPK pathway Technological Tools That Can Help the comprises a 3-layer kinase module, in which each element Understanding of Growth: is activated by serial phosphorylations on tyrosine and A Spotlight on Proteomics threonine residues and inactivated by dephosphorylation Among oncological research on the use of genomic tools by specific phosphatases.29 during the past decade, there has been a strong drive Angiogenesis is also fundamental to cancer growth toward proteomics. Proteomics indicates proteins expressed and , and it is regulated by many endogenous by a genome and is the systematic analysis of protein proangiogenic and antiangiogenic factors, such as vascular profiles of tissue, at a specific time point.39 Differentiated endothelial growth factor and its receptors.30 This process protein expression identification permits the association of includes degradation of the extracellular matrix, endo- these peptides with different physiologic events that have thelial cell proliferation, migration, and assembly of happened in the cells, tissue, and organs.40 endothelial cells into higher order structures.31 Many cancer biomarkers are the manifestation of Malignant cells from HNSCC evade immune recog- differences in post-transcriptional splicing, post-transla- nition and inhibit or exploit antitumor immune defenses. tional modifications, or both. Thus, proteomic tools are In this way, these cells escape from immune-mediated increasingly used in the postgenomic era to discover new destruction. Patients with HNSCC showed reduced cancer biomarkers (Fig. 1).41 Proteomics is mainly based þ þ peripheral blood concentrations of CD3 , CD4 , and on protein separation using 2-dimensional (2D) differen- þ CD8 T cells, which might persist even several years after tial in-gel electrophoresis. Two-dimensional gels provide curative .32 Many mechanisms for immune high-resolution expression, profiling thousands of evasion have been proposed, including escape from proteins from 1 tissue sample.42 The differential in-gel immune recognition and elimination caused by tumor electrophoresis technique is aimed at improving reprodu- factors,33 impaired T-lymphocyte activity, activity of cibility.43 To analyze the samples in differential in-gel immunosuppressive cells, and cytokine-mediating local electrophoresis, 2 pools of protein extracts are labeled and systemic effects.2 covalently with fluorescent cyanine dyes, Cy3 and Cy5, It has been postulated that Th2-like cytokines, like respectively. These labeled proteins are mixed and interleukin (IL)-434 and IL-13,35 in addition to others, separated in the same 2D gel, minimizing the reproduci- were responsible for HNSCC growth pattern and could bility problem.

4916 Cancer November 1, 2010 Head and Neck Cancer and Biomarkers/Rezende et al

Figure 1. Schematic figure shows head and neck (HNSCC) patient sample origin and main proteomic techniques used for studies. Numbers show the main head and neck cancer location: 1, paranasal sinuses; 2, nasal cavity; 3, oral cavity; 4, larynx; 5, pharynx. MALDI indicates matrix-assisted laser desorption/ionization; DIGE, differential in-gel electrophoresis; 2D, 2-dimensional; LC, liquid chromatography; iTRAQ, isobaric tag for relative and absolute quantitation; MS, mass spectrometry; ToF, time of flight.

Identification of proteins differentially expressed in have been used in breast, kidney, prostate, bladder, and the tumor samples can be performed using mass spec- human fibroblastic tumors in previous studies.48 Despite trometry (MS). Proteomic analysis can assist both in tissue heterogeneity, the protein expression in the disk the identification of biomarkers and proteins whose up- might not be representative for the entire tumor.49 regulation or down-regulation was perhaps instrumental These new technologies have been used successfully to the pathogenesis of the malignancy.44 to quantitatively and qualitatively characterize existing Furthermore, affinity bead-based purification has cancer markers in prostate cancer50 and to detect several been developed to make proteomic procedures suitable disease-associated proteins in tissue samples51 or complex for general MS analysis. Chemical chromatographic biological specimens, such as serum,52 cerebrospinal surfaces selectively purify certain subsets of proteins, fluid,51 nipple aspirates,53 pancreatic juice,54 saliva,55 and allowing unbound impurities to be removed by washing urine.56 Although these technologies have demonstrated with buffers. Proteins are typically enzymatically digested their suitability, they still provide only limited informa- after elution and before matrix-assisted laser desorption/ tion about the spatial variability of the cancer proteome. ionization (MALDI) time of flight (ToF) MS analysis, The protein composition of a malignant tumor varies among other forms of MS.45 MALDI-ToF MS sensitively qualitatively and quantitatively. This stems from the and precisely separates target proteins according to their intrinsic heterogeneity of solid tumors, the local inflam- mass-dependent velocities (m/z). Surface enhanced laser matory response, neovascularization, the development of desorption/ionization (SELDI) ToF MS uses chip-based a desmoplastic stroma, and so forth.57 A new approach to protein sample arrays with different chemical increase the diagnostic specificity and to characterize the chromatographic surfaces to selectively bind proteins cancer proteome profile in situ is MALDI imaging, which with specific chemical properties, such as hydrophobic, allows the detection of proteins in the tissue section and cationic-anionic, or metal-binding molecules, whereas the analysis of their spatial distribution without pre- nonspecifically bound proteins or impurities are removed fractionation.58 It allows an immediate evaluation of the by washing with buffers.46 Because SELDI ToF MS ana- putative significance of a biomarker, that is, the mass of lysis rapidly screens many samples at a time, it may be suita- interest can be allocated to histoanatomical structures ble for clinical use and for novel biomarkers.47 such as tumor cells, tumor stroma, tumor vessels, and Tissue arrays, another important technique, allow inflammation. the simultaneous in situ analysis of many tumors on the DNA, RNA, and protein levels using in situ hybridization HNC Proteomic Findings or immunohistochemistry.48 This technique can provide Even after some cancer findings, the survival rates for an efficient, high-throughput method for evaluation of patients with HNSCC have remained unchanged over the protein expression in large cohort studies using archival past 30 years.59 Prognosis and treatment are influenced by formalin-fixed, paraffin-embedded tissue. Tissue arrays the stage of disease at diagnosis. Patients with HNSCC

Cancer November 1, 2010 4917 Review Article often present with advanced stage disease, which is asso- superoxide dismutase 2. In the following year, this ciated with a poorer prognosis and requires more aggres- group selected 1 of the above up-regulated proteins— sive therapy, which in turn results in increased functional stathmin—to correlate its presence with tumor progres- disability.60 sion and poor prognosis.64 They evaluated the state of Research efforts directed at improving early detec- stathmin protein and mRNA expression in OSCC- tion of malignancy have focused on the identification of derived cell lines and human primary OSCC. A signifi- genetic alterations and tumor biomarkers. Conventional cant increase in stathmin expression was reported in all diagnostic techniques, including direct inspection or examined OSCC-derived cell lines compared with human increasingly sophisticated imaging technology, such as normal oral keratinocytes. In immunohistochemistry, positron emission tomography-computer tomography, 65% of the OSCC was positive for stathmin, and no are limited in their ability to detect HNSCC at its earliest immunoreaction was observed in corresponding normal stages and are ineffective for use as a screening tool in tissues. Moreover, stathmin expression status was corre- high-risk populations.61 Biomarkers associated with lated with the stage of TNM , and a statistical HNSCC have revealed considerable variety in protein correlation was found between protein expression status expression, and as a consequence, there is a variety of and disease-free survival. These results suggested that the potential biomarkers (Table 1). These potential bio- expression of stathmin could contribute to cancer pro- markers present different biochemical functions to gain gression/prognosis, and that stathmin may have potential their antitumor activity. However, the current literature as a biomarker and a therapeutic target for OSCC. These emphasizes the major abundant proteins, whereas little results were in accordance with the high level of stathmin information is available for less abundant proteins that expression in many types of cancer, including can be the key to discovering HNSCC markers. Another and ,65 prostate carcinoma,66 ovarian carci- important point is that the study of HNC involves differ- noma,67 Wilms tumor,68 breast carcinoma,69 and adenoid ent tissues and systems, so the search for specific bio- cystic carcinoma of the salivary glands.70 markers must be related to each tissue. These reasons are To identify potential biomarkers for laryngeal SCC, probably related to the finding that until now, these Sewell et al compared the protein profile of laryngeal potential biomarkers are not ready to be clinically used. cancer tissue with normal mucosal samples using 2D gel electrophoresis and MS.44 The differentially expressed Tissue Biomarker Studies proteins were stratifin, S100 calcium-binding protein Differential in-gel electrophoresis technique was used for A9, p21-ARC, stathmin, and enolase. Several of these the identification of esophageal squamous cell cancer-spe- proteins regulate cellular proliferation, differentiation, cific protein markers by Zhou et al.62 Both esophageal and apoptosis, and their function may directly relate to cancer and normal squamous epithelium cells were pro- the pathogenesis of cancer in these patients. These find- cured from the same esophageal tumor sample by laser ings revealed potential biomarkers that may contribute to capture microdissection.62 2D gel images and MS were the pathogenesis of laryngeal carcinoma, and that may be performed, and a down-regulation of protein annexin I suitable as targets for novel therapeutic and/or diagnostic and an up-regulation of tumor rejection in esoph- modalities. However, because of the small sample size, ageal squamous cell cancer were observed. This result these findings cannot be generalized for all laryngeal carci- demonstrates that the 2D differential in-gel electrophore- noma cases, but they provided a scientific basis for further sis technique, in combination with MS, is a powerful tool proteomic analyses of laryngeal cancer samples. for the molecular characterization of cancer progression The identification of and identification of cancer-specific protein markers. biomarkers was observed in Cheng et al’s study.71 After Koike et al performed proteomic profiling between protein separation by 2D gel electrophoresis and MS human normal oral keratinocytes and OSCC-derived cell identification, the expression levels of stathmin, 14-3-3r, lines (HSC-2 and HSC-3) using fluorescent 2D differen- and annexin I in the 2 types of tissues were confirmed and tial in-gel electrophoresis and MALDI ToF peptide mass related to differentiation degree and/or metastatic poten- fingerprinting.63 Some of the differential proteins tial of the nasopharyngeal cell lines. Significant stathmin between the groups included annexin A1, heat shock up-regulation and down-regulation of 14-3-3r and protein 27, lamin A/C, interleukin 1 receptor antagonist, annexin I were observed in nasopharyngeal carcinoma serine-proteinase inhibitor clade B5, stathmin 1, and versus normal nasopharyngeal epithelial tissue, and

4918 Cancer November 1, 2010 Cancer oebr1 2010 1, November

Table 1. Potential HNSCC Diagnostic Biomarkers Sample Potential Biomarker Biochemistry Function Technology Used

Tissue Annexin Ia,b Epithelial differentiation and growth regulation DIGEþMS/2DEþMS þ Annexin A1c Ca2 -dependent phospholipid-binding proteins 2DEþMALDI ToF MS Heat shock protein 27c Immune response protein 2DEþMALDI ToF MS Lamin A/Cc Nuclear stability, chromatin structure, and gene expression protein 2DEþMALDI ToF MS Interleukin 1 receptor antagonistc Immune response protein 2DEþMALDI ToF MS Serine proteinase inhibitor clade B5c Protease 2DEþMALDI ToF MS Stathmin 1b,c,d,e Cell motility, migration, and microtubule dynamics protein 2DEþMALDI ToF MS Superoxide dismutase 2c Enzyme 2DEþMALDI ToF MS Stratifine p53-regulated inhibitor of G2/M progression 14-3-3 sigma 2DEþMS S100 calcium-binding protein A9e Cell cycle regulator protein 2DEþMS P21-ARCe Actin related protein 2DEþMS Enolasee Phosphopyruvate dehydratase enzyme 2DEþMS 14-3-3rb Cellular differentiation proteins in epithelial tumors and transcription 2DEþMS of c-myc protein inhibition Cytokeratinsf Cytoskeletal proteins Tandem MSþbioinformatic analysis Intermediate filament proteinsf Cytoskeletal proteins Tandem MSþbioinformatic analysis YWHAZg Mediate signal transduction protein LCþtandem MS hnRNPKg Heterogeneous nuclear ribonucleoprotein K LCþtandem MS al et Biomarkers/Rezende and Cancer Neck and Head Plasma Fibrinogen a-chain fragmenth Cellular adhesion, proliferation, and migration of protein during MALDI ToF/ToFþmascot identification carcinogenesis Ig gamma-3 chain C regioni Immune response protein Nano-LC ESI MS/MSþmascot identification Complement component C4ai Immune response protein MALDI ToF/ToFþCID and LIFT acquired MS/MS Ig kappa chain C regioni Immune response protein Nano-LC ESI MS/MSþmascot identification Chromiumi Trace element ICP-MS Serum HSP 70j Immune response protein 2DEþMALDI ToF MS sICAM1j Intercellular adhesion molecule 2DEþMALDI ToF MS SAAj Acute-phase protein 2DEþMALDI ToF MS

HNSCC indicates head and neck squamous cell carcinoma; DIGE, differential in-gel electrophoresis; MS, mass spectrometry; 2DE, 2-dimensional electrophoresis; MALDI, matrix-assisted laser desorption/ ionization; ToF, time of flight; LC, liquid chromatography; Ig, immunoglobulin; ESI, electrospray ionization; CID, collision-induced dissociation; ICP, inductively coupled plasma; LIFT, laser-induced fragmenta- tion tandem. a Zhou 200262 ; b Cheng 200871 ; c Koike 200563 ; d Kouzu 200664 ; e Sewell 200744 ; f Patel 200872 ; g Ralhan 200973 ; h Cheng 200545 ; i Gomes 201079 ; j Liao 2008.80 4919 Review Article significant down-regulation of 14-3-3r and annexin I was nificant differences in protein expression between differ- also observed in metastasis versus primary ent subsites and proposed that the observed heterogeneity nasopharyngeal carcinoma. In addition, stathmin up- may reflect divergent etiologic pathways irrespective of regulation and down-regulation of 14-3-3r and annexin I subsite.74 were significantly correlated with poor histological differ- Patel et al used imaging MS with chemical inkjet entiation, advanced clinical stage, and recurrence, whereas printing, revealing differential protein expression in down-regulation of 14-3-3r and annexin I was also human OSCC.75 The analysis of the resulting protein significantly correlated with lymph node and distant profiles reveals spectral features at 4500 and 8360 Da metastasis. Survival curves showed that patients with that strongly correlate with the SCC region of the tongue. stathmin up-regulation and down-regulation of 14-3-3r The feature selection of this study was only performed on and annexin I had a poor prognosis. These data suggest a single patient, and then the selected peaks were tested on that these proteins are potential biomarkers for the differ- a further 3 patients, which gives some confidence in these entiation and prognosis of nasopharyngeal carcinoma. diagnostic peaks. However, the identity of these 2 proteins By using a different form of HNSCC sample, Patel is still unknown. According to the authors, these studies et al described the utility of a novel proteomics platform needed to be extended to a larger sample set that includes for the global detection of expressed proteins in formalin- premalignant lesions, and over a longer period, but it fixed paraffin-embedded tissue.72 This approach enabled demonstrated that this MS profiling technique produced identification of a large number of molecules, including reproducible, informative chemical images for clinical cytokeratins and intermediate filament proteins, differen- pathology. tiation markers, proteins involved in stem cell mainte- nance, signal transduction, and cell cycle regulation, Plasma and Serum Biomarker Studies growth and angiogenic factors, matrix-degrading In addition to tumor tissues, plasma and serum from proteases, and proteins with tumor suppressive and onco- patients could be alternative sources for the study of dif- genic potential. The ability to correlate protein expression ferential expression, especially for proteomic approaches. profiles with histopathologic classification of disease may Cheng et al observed the high specificity and sensitivity of allow the development of novel biomarkers of diagnostic the fibrinogen a-chain fragment in oral cancer plasma, and prognostic value and may help identify novel targets suggesting that it may be a clinically useful tumor for therapeutic intervention in HNSCC. marker.45 In this study, the fibrinogen fragment presented By using online liquid chromatography and tandem higher sensitivity (100%) and specificity (97%) for cancer MS, Ralhan et al compared isobaric mass tags (iTRAQ)- than the other markers detected, by MALDI ToF/ToF labeled oral and normal tissues against pooled followed by Mascot identification. Previous studies in ani- normal control.73 Three best-performing biomarkers mal models have shown that inhibition of fibrinogen were identified by iTRAQ analysis and verified by strongly diminishes the development of metastatic immunohistochemistry: stratifin, YWHAZ, and hnRNPK. cancer, further demonstrating the important role of fibri- These biomarkers pointed to some key regulatory proteins nogen in sustaining invasion and survival of tumor cells.76 that link inflammation and development of epithelial In the series of coagulation markers tested, only the dysplasia in oral premalignant lesions, because they discrim- fibrinogen a-chain was significantly increased in gastric inate dysplasias from normal tissues. Pathway analysis cancer, and it was correlated with tumor TNM stages.77 revealed direct interactions between all 3 biomarkers and Similarly, the fibrinogen a-chain peptide has been found their involvement in 2 major networks involved in inflam- to be significantly increased in .78 mation, signaling, proliferation, regulation of gene expres- Our group in a previous study analyzed plasmas of sion, and cancer. patients with laryngeal cancer and of healthy smokers by Tissue microarray and 2D differential in-gel electro- 2D gel electrophoresis and MS. Few differences were phoresis were used to evaluate HNSCC for differences found between cancer and control patients. However, 3 in protein expression between oral cavity, oropharynx, spots gathered between platelet count increment 7.3 and larynx, and hypopharynx subsites. For the tissue array 7.6 with different molecular masses appeared exclusively study, the chosen proteins were cyclin D1, p53 Rb, and in cancer profiles. From 10 spots identified, 6 correspond p14, and for the 2D differential in-gel electrophoresis, to components, including the 3 differ- total protein was extracted. The authors did not find sig- ential ones: Ig gamma-3 chain C region, complement

4920 Cancer November 1, 2010 Head and Neck Cancer and Biomarkers/Rezende et al

Table 2. Potential HNSCC Physiology Biomarkers Function Regulated Protein Technology Used

Hypoxiaa IjB kinase b 2DEþpowerblot Tumor transitionb Multiplex tissue immunoblotting Early events CK-4, CK-14, annexin 1 Normal to dysplasia Cox-2, P53 Pro-oncogenesc Transcription factors MALDI ToF MS Nuclear receptors Enzymes MMP-9 activationd TNF-a 2DEþMS Tissue differentiation degree/metastatic Cathepsin D 2DEþMS potentiale

HNSCC indicates head and neck squamous cell carcinoma; 2DE, 2-dimensional electrophoresis; MALDI, matrix-assisted laser desorption/ionization; ToF, time of flight; MS, mass spectrometry; MMP, matrix metalloproteinase; TNF, tumor necro- sis factor. a Chen 200481; b Chung 200684; c Koehn 200885; d Hohberger 200886; e Cheng 2008.88 component C4a, and Ig kappa chain C region. Because Hypoxia is a major determinant of local, regional, and tobacco is the main cause of laryngeal cancer, and it distant failure after anticancer therapy.82 At the molecular contains various carcinogenic components, including level, hypoxia selects tumors with an increased malignant metallic elements, the presence of these elements was stud- phenotype,83 resulting in resistance to apoptosis and ied. Several trace elements in the identified proteins were greater propensity for distant metastases. In this way, the determined by inductively coupled plasma MS, where identification of hypoxia markers may influence the chromium was increased in all proteins analyzed from choice of therapeutic modality. By using 2D gel electro- patients with cancer. Our results reinforce the importance phoresis and powerBlot (-based array), they of the immune response as a target in the understanding identified a group of 20 proteins. The majority of these and treatment of laryngeal cancer and the possibility that proteins, such as IjB kinase b, MKK3b, highly expressed chromium is important in the carcinogenic progress.79 in cancer, density-regulated protein 1, P150, nuclear In a similar way, serum biomarkers were also used transport factor 2, binder of ARL2, paxillin, and transcrip- for HNSCC diagnosis. Liao et al analyzed serum tion termination factor I have not been previously reported to proteome for profiling protein markers associated with be hypoxia inducible. A strong correlation between IjB carcinogenesis and lymph node metastasis in nasopharyn- kinase b protein expression and tumor oxygenation was geal carcinoma.80 By 2D image analysis, MALDI ToF observed. They found that IjBkinaseb is induced by MS identification, and enzyme-linked immunosorbent hypoxia and plays an important role in mediating cell survival assay validation, 3 proteins were selected. The overexpres- under hypoxic stress. In human HNSCC, expression of this sion of HSP70, sICAM-1, and SAA was observed in protein appeared to be a good indicator of tumor hypoxia nasopharyngeal carcinoma patients and may be of great and may represent a novel anticancer therapeutic target.81 underlying significance in the clinical detection and Studying the normal to tumor transition of eso- management of this kind of cancer. phageal SCC, Chung et al quantified the change in 7 In summary, analysis of the resultant protein pro- proteins in this tissue transition using multiplex tissue file may have greater utility in early diagnosis by selecting immunoblotting.84 Their data suggest that decreased ex- a combination of protein alterations rather than by fo- pression of pan-cytokeratin (CK)-4, CK-14, and annexin cusing on specific tumor markers, which may vary 1 are early events. In contrast, the modest increase between individual patients. observed in cyclooxygenase-2 and p53 protein expression with progression from normal to dysplasia suggests that HNC Physiology Biomarker Studies these markers may be most informative in more The proteomic findings in HNC are not limited to dis- advanced neoplasia. The increase in cysteine (secreted covering diagnostic biomarkers but also include discover- protein acidic and rich in cysteine) even in stroma under- ing targets for tumor-specific therapeutic modalities lying dysplasia, as well as the potential to measure the (Table 2). Chen et al identified the hypoxia-regulated pro- protein in serum, makes it a potential biomarker of early teins in HNC by proteomic and tissue array profiling.81 disease. These findings suggest that changes in protein

Cancer November 1, 2010 4921 Review Article expression can be detected during the transition to dys- The expression level of cathepsin D in the 2 types of plasia and may be useful biomarkers. tissues was confirmed by Western blotting and related to Koehn et al analyzed by 2D gel electrophoresis, differentiation degree and metastatic potential of the followed by MALDI ToF MS, subcellular fractions from nasopharyngeal carcinoma cell lines. Down-regulated OSCC and corresponding control samples, enriched in cathepsin D expression by small interfering RNA signifi- mitochondrial and cytosolic proteins, as well as blood cantly decreased the in vitro invasive ability of cancer cells. from the tumor.85 They identified 350 different gene Significant cathepsin D down-regulation was observed in products, 20 proteins with deranged levels in the cancer nasopharyngeal carcinoma versus normal nasopharyngeal samples, of which 16 were up-regulated. Eight of the epithelial tissues, whereas significant cathepsin D up-reg- up-regulated proteins are associated with gene products ulation was observed in lymph node metastasis versus involved in the pathways of proto- p53, MYC, primary nasopharyngeal carcinoma. In addition, cathep- and MYCN, which are transcription factors, nuclear sin D down-regulation was significantly correlated with receptors, or enzymes or have other functions. These poor histological differentiation, whereas cathepsin D results indicated that proteomic analysis is a powerful tool up-regulation was significantly correlated with advanced in systems biology for the elucidation of the complexity of clinical stage, recurrence, and lymph node and distant expression profiles in cellular processes. metastasis. Furthermore, survival curves showed that Elucidating factors responsible for oral cancer pro- patients with cathepsin D up-regulation had a poor prog- gression at the late preneoplasia/invasion interface were nosis. These findings could have clinical value in distin- investigated by Hohberger et al, who examined the inte- guishing histological grades, predicting the prognosis of gral role of matrix metalloproteinase (MMP) activation at nasopharyngeal cancer, and identifying nasopharyngeal this interface.86 These MMPs allow degradation of the cancer patients who are at high risk of metastasis and extracellular matrix on secretion.87 In keratinocytes and recurrence. other cells, they have been associated with tissue remodel- ing processes, including embryogenesis, tumor invasion Conclusive Remarks and Future Directions and metastasis, wound healing, and inflammatory Proteomics, together with genomics, is well on the way to processes.87 The authors believe that inflammation may molecular characterization of the different tumor types be a key factor in oral cancer progression in late preneo- and to detecting diagnostic and novel therapeutic targets plasia, and that the inflammation-associated transcription for disease treatment. In addition, functional proteomics factor, nuclear factor-jB, may be the principal regulator methods have also been developed to study the intracellu- of these processes. In this study, they discovered that lar signaling pathways that underlie the development of the MMP-9 promoter was significantly stimulated by phor- cancers, which could therefore significantly contribute to bol myristate acetate and tumor necrosis factor (TNF)-a on the efficient performance of the entire discovery process. luciferase reporter gene assays. Furthermore, functional Potential areas for improvement of this approach to MMP-9 promoter activation was accompanied by signifi- HNSCC will arise from studies that include high-risk cant increases in MMP-9 gene expression. Functional activa- groups as well as the analysis of serum proteomic profiles tion of the MMP-9 protein was simulated by TNF-a and before, during, and after definitive treatment of HNSCC phorbol myristate acetate on a fluorescent enzyme-linked to determine whether this technique can be equally useful immunosorbent serologic assay. Finally, they observed that for monitoring patients for persistent or recurrent disease. MMP-9 is the third most significant protein in saliva of oral In addition, because prognosis and treatment are influ- cavity cancer patients over normal controls. These results enced by early cancer diagnosis, the determination of a suggest that TNF-a has the capacity to provide stimulation cancer biomarker at a proteome level allows this evalua- of events related to early invasion of oral cavity cancer.86 tion to be faster. Trying to identify biomarkers for differentiation and prognosis of nasopharyngeal carcinoma by proteomic CONFLICT OF INTEREST DISCLOSURES analysis, Cheng et al analyzed proteins from pooled Supported by Universidade Catolica de Brasilia (UCB), Con- microdissected nasopharyngeal carcinoma and normal selho Nacional de Desenvolvimento Cientı´fico e Technolo´gico nasopharyngeal epithelial tissues.88 Thirty-six differential (CNPq), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Fundac¸a˜o de Amparo a Pesquisa de proteins between the nasopharyngeal carcinoma and Minas Gerais (FAPEMIG), and Fundac¸a˜o de Amparo do Dis- normal nasopharyngeal epithelial tissues were identified. trito Federal (FAPDF).

4922 Cancer November 1, 2010 Head and Neck Cancer and Biomarkers/Rezende et al

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