www.clinical.proteomics-journal.com
Page 1
Proteomics - Clinical Applications
Title page Title Potential biomarkers for oral squamous cell carcinoma: proteomics discovery and clinical validation Yan-hong Ni, Liang Ding, Qin-gang Hu*, Zi-Chun Hua* Nanjing Stomatological Hospital and the State Key Laboratory of Pharmaceutical Biotechnology, School of Stomatology, Nanjing University, Nanjing 210008, P.R. China
Corrosponding author Qin-Gang Hu, PhD, MD, Department of Oral and Maxillofacial Surgery, Institute and Hospital of Stomatology, Nanjing University Medical School, Nanjing Stomatological Hospital, 30 Zhong Yang Road, Nanjing 210008, P. R. China Tel.: +86 025 83620101 Fax: +86 025 83357174. E-mail: [email protected]
Zi-chun Hua, PhD Nanjing Stomatological Hospital and the State Key Laboratory of Pharmaceutical Biotechnology, School of Stomatology, Nanjing University, Nanjing 210008, P.R. China Tel.: +86 025 83620101 Fax: +86 025 83357174. E-mail: huazc @nju.edu.cn
Received: 31-Jul-2014; Revised: 23-Oct-2014; Accepted: 24-Nov-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/prca.201400091. This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 2
Proteomics - Clinical Applications
Abbreviations OSCC
Oral squamous cell carcinoma
FFPE
formalin-fixed, paraffin-embedded
IHC
immunohistochemistry
MS
Mass Spectrometry
SELDI-TOF-MS surface-enhanced laser-desorption–ionization time-of-flight mass spectrometry 2DICAL
2-dimensional image-converted analysis of liquid chromatography and mass spectrometry
Abstract Oral squamous cell carcinoma (OSCC) is the worldwide concerned cancer. In spite of the advances in treatment, the 5-year survival rate has only increased subtly during the past two decades, which is largely due to the advanced stages of disease at diagnosis and the frequent development of relapse and second primary tumors. Therefore, the identification of underlying OSCC protein biomarker during cancer initiation and progression could aid the diagnosis and treatment of OSCC. In this review, recent researches on proteomics analysis of tissue, salivary and serum for early detection and evaluation aggressiveness and occurrence of OSCC were summarized. The emphasis is placed on early detection by tissues, salivary and serum of patients with histologically defined OSCC patients. Although lots of researches for searching OSCC protein biomarker have done, few common protein biomarkers have been detected. Low-redundant protein in tissues, salivary and serum from OSCC may more accurately reflected the progression of OSCC, so novel approach for the depth research strategy and the sample choice for proteomics are of importance in OSCC biomarker discovery.
This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 3
Proteomics - Clinical Applications
Key words oral squamous cell carcinoma (OSCC); proteomics; tumor biomarkers Total number of words: 8847
1 Introduction 1.1 Oral squamouse cell carcinoma characteristics Squamous cell carcinoma is the most common histological type of head and neck cancer, accounting for 90% of all head and neck malignancies[1]. Oral squamous cell carcinoma (OSCC) is a significant cancer worldwide, and
ranks as the eighth most common cancer in males with an average global
incidence of 6.3 per 100,000[1]. Although several therapeutic regimens, including surgery, radiotherapy, and chemotherapy, have been integrally applied for three decades, the 5-year survival rate has only been increasing subtly during the past two decades[2]. Moreover, it is more prevalent in developing countries than in developed countries. This figure is largely due to the advanced stages of the disease at diagnosis which, in turn, limited proper treatments. So far, the most important risk factors identified for OSCC are tobacco use and alcohol consumption, which seem to have a synergistic effect. Recently, it has reported that persistent infection with human papillomavirus (HPV) is also a major risk factor for the development of OSCC, in particular for oropharyngeal squamous cell carcinoma (OPSCC) including tonsillar cancers and base of tongue cancer[3-5]. OSCC follows a similar pattern in its development, and is preceded by precancerous lesions. The malignant potential of oral leukoplakia (OLK), which is the most common type of precancerous type, This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 4
Proteomics - Clinical Applications
was evidenced by the progression from low grade dysplasia, high grade dysplasia, and ultimately to invasive carcinoma. The prognosis for patients with OSCC is largely determined by the stage at presentation and the extent of the tumor, as well as the presence of lymph-node metastases and distant metastases.. Staging of OSCC is determined by clinical examination, imaging, cytology of lymph nodes and definite histopathology after surgery[6]. Disappointingly, survival has not markedly improved in recent decades because patients still frequently develop local-regional recurrences, distant metastases and second primary tumors. The limitedly available information on the molecular carcinogenesis of OSCC or the genetic and biological heterogeneity of the disease has hampered the diagnosis and therapeutic strategies for the development of OSCC. 1.2 The role of biomarker in diagnosis and prognosis of OSCC Patients with early stage OSCC (stage I or II) and non-nodal metastases, who have been treated by surgery or radiotherapy, have excellent 5-year survival rate.However, two thirds of all OSCC patients present with the advanced stage (stage III or IV), due to the fact that large majority of OSCCs arise de novo, without preceding visible changes in the mucosa. Additionally, OSCC may progress from early to advanced stages within a very short time perhaps a few weeks, which hamper early diagnosis for OSCC. During the last decades, progress has been made in the efforts for searching novel treatment modalities for OSCC[7, 8]. Despite these developments also resulting in improved local-regional control and reduced treatment-related morbidity, 5-year survival rate has not improved significantly[9]. Because early detection represents one of the most promising approaches to reducing the growing cancer burden[10], so early diagnosis of primary tumors remains the key to improve treatment outcome. Therefore, deeper
This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 5
Proteomics - Clinical Applications
understanding of the molecular biology of OSCC can provide new insights into its mechanisms of development and progression and it also can provides various biomarkers with a potential application for cancer screening. Alterations in gene sequences, expression levels and protein structure or function have been associated with every type of cancer. These ‘molecular markers’ can be useful in detecting cancer, determining prognosis and monitoring disease progression or therapeutic response[11]. Biomarkers not only can be a single molecular but also a series of molecular，which can be found in a variety of fluids, tissues and cell lines. Biomarkers can be DNA, mRNA, proteins, metabolites, or processes such as apoptosis, angiogenesis or proliferation[12]. The cancer biomarker, which is produced either by the tumor itself or by other tissues, is a molecular signature indicating the physiologic and pathologic changes in a particular tissue or cell type during the development of cancer. There are several types of biomarkers, including diagnostic biomarker, prognostic biomarker and stratification biomarker, which is also be regarded as predictive biomarker. The reported first cancer marker was the light chain of immunoglobulin in the urine, as identified in 75% of patients with myeloma. For example, CA-125 which has been proved to be an prognostic biomarker for ovarian epithelial cancer[14], carcinoembryonic antigen (CEA) which has been proved to be the prognosis biomarker for colon cancer and prostate-specific antigen (PSA) which has been considered to be one of the best prostate cancer markers[15, 16]. Genomic and proteomic technologies have greatly increased the biomarker number and it has been realized that multi-parametric analysis of genes or proteins is especially important for searching biomarkers. mRNA information could not best reflect the function of proteins, which are the functional components, the use of proteomic patterns to enable tumor diagnosis seems to be much more promising.
This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 6
Proteomics - Clinical Applications
Proteins produced by cancer cells or their microenvironment may eventually enter the circulation system and the expression patterns of these proteins could be assessed by mass spectrometry. Proteomics can help us to deeply understand the changes in the levels of multiple proteins involved in oncogenesis and cancer progression and identify new diagnostic or prognostic biomarkers [17]. Mass-spectrometry-based methods of proteomic analysis have improved and include more-advanced technology that brings higher mass accuracy, higher detection capability, and shorter cycling times, thereby enabling increased throughput and more-reliable data[18]. Technologies such as differential in-gel electrophoresis, two-dimensional polyacrylamide gel electrophoresis and multidimensional protein-identification technology can be used for high-throughput protein profiling. The technology that has received considerable attention involves the use of a minute amount of unfractionated serum sample added to a 'protein-chip', which is subsequently analyzed
by
surface-enhanced
laser-desorption–ionization
time-of-flight
mass
spectrometry
(SELDI-TOF-MS) to generate a proteomic signature of serum[19]. Quantitative proteomics is an important branch of proteomics that is applied to quantify and identify all the proteins expressed by a whole genome or in a complex mixture. Comparative proteomics, a study of profiling the global protein expressions within a cell, tissue or organism in comparison to its control, has become one of the promising approaches for the discovery of candidate disease biomarkers.
2 OSCC biomarkers and proteomics The identification of molecular mechanisms underlying cancer initiation and progression could aid in the development of new diagnostic and treatment options for the disease. The processes of cancer cell transformation and progression is extremely complex events involving the deregulation of a variety of genes which controlled cell proliferation, differentiation and cell death. Different proteins, probably in the This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 7
hundreds or thousands, may be up-regulated or down-regulated
Proteomics - Clinical Applications
simultaneously and may conduct
distinct cell functions. Therefore, the analysis of hundreds of proteins simultaneously holds great promise for accurately predicting the function of marker proteins. 2.1. Tissue proteomics analysis for OSCC All the researches on detecting OSCC biomarkers from OSCC tissues by proteomics had been summarized in Table 1. 2.1.1 Protein biomarkers for early detection of OSCC There are fewer researches about discrimination the protein expression profiles between tumor tissue and precancerous lesion with different stages of dysplasia. In the process of disease progress from OLK to OSCC, some underlying molecular signaling network must have been changed. Therefore, in order to identify novel diagnostic targets underlying the malignant transformation from precancerous to cancerous tissue, Chen et al. evaluated protein expression differences by proteomics methods. Six proteins obtained from tumors and OLK tissues with dysplasia were analyzed by two-dimensional gel electrophoresis, followed by ESI-Q-TOF tandem mass spectrometry, then Gene Ontology (GO) analysis and biological networks were applied to identify new therapeutic targets or protein markers. Results identified approximately 85 differentially and constantly expressed proteins, including 52 up-regulated and 33 down-regulated. Three proteosome activators PA28a, b and g were shown to have up-regulated in OSCC cells relative to oral keratinocytes indicating three homologs of PA28 may play an important role in malignant transformation[20]. In order to identify the proteins that are differentially expressed in OSCC compared to adjacent non-tumor tissue, Pitak et al. analyzed fresh tissue samples of cancer and their adjacent normal mucosa from 10 patients with well differentiated OSCC. Five cases were stage I and II (early-stage group) and five This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
cases
were
stage
III
and
IV
Page 8
(late-stage
group).
Proteomics - Clinical Applications
Two-dimensional
(2D)
sodium
dodecyl
sulphate–polyacrylamide gel electrophoresis accompanied by mass spectrometry (matrix-assisted laser desorption/ioni-sation-time-of-flight mass spectrometry and liquid chromatography–tandem mass spectrometry) was used to analyze and identify the differentially expressed proteins. A total of 68 proteins (63 up-regulated, five down-regulated) were differentially expressed in early-stage disease, and 39 proteins (37 up-regulated, two down-regulated) were significantly altered in late-stage disease. Among these, 14 proteins were altered in both groups. A total of 44 proteins were identified, including heat shock proteins (HSPs: Hsp90, HSPA5 and HSPA8), keratins (K1, K6A and K17), tubulin, cofilin 1, 14-3-3s and metabolic enzymes. These proteins are involved in various cellular processes essential for cell growth, survival and cell migration[21]. In order to unveil the molecular signals triggering the onset of oral carcinogens, two-dimensional gel electrophoresis method was used to analyze 10 pairs of tissues T3 (9 N0, 1 N2) including primary OSCC and their matched adjacent normal surrounding mucosa specimens. 41 proteins were identified as commonly over-expressed in OSCC tissues. In OSCC tissues, alphaB-crystallin, tropomyosin 2, myosin light chain 1, HSP27, stratifin, thioredoxin-dependent peroxide reductase, flavin reductase, vimentin, rho GDP-dissociation inhibitor 2 (rho GDI-2), glutathione S-transferase Pi (GST-pi) and superoxide dismutase [Mn] (MnSOD) were significantly over-expressed (an average of 7.2, 6.0, 5.7, 4.3, 3.6, 3.4, 3.0, 3.0, 2.6, 2.5, 2.1-fold, respectively)[22].
Tumor tissues are the reliable sources for searching tumor biomarker. Formalin fixation and tissue embedding in paraffin wax (FFPE) is a universal approach for tissue processing, histologic evaluation, and routine diagnosis, as it preserves the cellular morphology and tissue architecture. Combination of
This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 9
Proteomics - Clinical Applications
proteomic analysis with laser capture microdissection may afford performing the proteomic characterization of normal and pathologic cell populations from clinical specimens, thus reflecting their protein make up as they exist in vivo. Therefore, researchers combined the use of laser capture micro-dissection methods enabling the extraction of peptides directly from FFPE samples, and optimized chromatographic approaches to undertake a large-scale proteomic study to identify proteins expressed in FFPE head and neck squmouse cell carcinoma (HNSCC) tissues. Appropriate paraffin blocks of formalin-fixed tissues from 4 normal squamous epitheliums, and 4 well differentiated (WD), 4 moderately differentiated (MD), and 4 poorly differentiated (PD) HNSCC were retrieved to help identify novel molecular targets for the early detection, prevention, and treatment of HNSCC. A large number of proteins expressed in normal oral epithelium and HNSCC, including cytokeratins, intermediate filaments, differentiation markers, and proteins involved in stem cell maintenance, signal transduction, migration, cell cycle regulation, growth and angiogenesis, matrix degradation, and proteins with tumor suppressive and oncogenic potential, were readily detected. To enable the comparison of protein expression across all tissue samples, they used a spectral count method in which each protein that was identified in any one set was evaluated based on the number of unique peptides identified, and the total number of times that those peptides were observed in five successive runs for each set. The number of proteins identified for each set as a group (all four sample results pooled together) ranged from 391 in the normal oral epithelia to 866 total proteins in the WD HNSCC tumors. As indicated, 115 proteins were identified as shared across all normal and tumor tissues. Cytokeratins and desmosomal proteins, stood out as the most abundant in all four-tissue sets. Most keratins were nearly equally represented across all tissue samples, for example, cytokeratin 5, Cytokeratin 14, cytokeratin 4, desmoplakin, plakophilin 1, periplakin, and desmoglein
This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 10
Proteomics - Clinical Applications
precursors, were prominent in all samples. Also notable the family of calcium-binding proteins (calgranulin A, calgranulin B, S10 0 A14, calmod ulinlike protein), keratinocyte differentiation markers (involucrin, small pro-line-rich protein 3, profilin 1, cornifin A, cornifin B), and many membrane-related molecules (annexin A1, actin-like protein 2) were identified. A number of proteins including heat shock protein 27 (HSP27) (15), HSP70 (16), and glutathioneS -transferase (15), expected to be of higher abundance in tumor samples, were identified. A number of signaling molecules (e.g., Ras GTPase-activating-like protein IQGAP1, obscurin, tyrosine-protein kinase ITK) were also identified. Conversely, the DNA excision repair protein ERCC-5 was found to be less abundant in tumor samples. These data indicated that the analysis of FFPE tissues enables the identification of a broad range of functionally diverse proteins in normal and tumor squamous epithelia, albeit many exhibit a distinct expression profile. Forty-two proteins unique to HNSCC were readily detected by multiple peptides (>10). Among them, the most abundant was vimentin, a protein involved in epithelial-mesenchymal transition. A variety of proteins involved in cell migration, signaling, and proteolysis were also identified. Eighty-five less abundant proteins (i.e., identified by >4 peptides but
Page 1
Proteomics - Clinical Applications
Title page Title Potential biomarkers for oral squamous cell carcinoma: proteomics discovery and clinical validation Yan-hong Ni, Liang Ding, Qin-gang Hu*, Zi-Chun Hua* Nanjing Stomatological Hospital and the State Key Laboratory of Pharmaceutical Biotechnology, School of Stomatology, Nanjing University, Nanjing 210008, P.R. China
Corrosponding author Qin-Gang Hu, PhD, MD, Department of Oral and Maxillofacial Surgery, Institute and Hospital of Stomatology, Nanjing University Medical School, Nanjing Stomatological Hospital, 30 Zhong Yang Road, Nanjing 210008, P. R. China Tel.: +86 025 83620101 Fax: +86 025 83357174. E-mail: [email protected]
Zi-chun Hua, PhD Nanjing Stomatological Hospital and the State Key Laboratory of Pharmaceutical Biotechnology, School of Stomatology, Nanjing University, Nanjing 210008, P.R. China Tel.: +86 025 83620101 Fax: +86 025 83357174. E-mail: huazc @nju.edu.cn
Received: 31-Jul-2014; Revised: 23-Oct-2014; Accepted: 24-Nov-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/prca.201400091. This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 2
Proteomics - Clinical Applications
Abbreviations OSCC
Oral squamous cell carcinoma
FFPE
formalin-fixed, paraffin-embedded
IHC
immunohistochemistry
MS
Mass Spectrometry
SELDI-TOF-MS surface-enhanced laser-desorption–ionization time-of-flight mass spectrometry 2DICAL
2-dimensional image-converted analysis of liquid chromatography and mass spectrometry
Abstract Oral squamous cell carcinoma (OSCC) is the worldwide concerned cancer. In spite of the advances in treatment, the 5-year survival rate has only increased subtly during the past two decades, which is largely due to the advanced stages of disease at diagnosis and the frequent development of relapse and second primary tumors. Therefore, the identification of underlying OSCC protein biomarker during cancer initiation and progression could aid the diagnosis and treatment of OSCC. In this review, recent researches on proteomics analysis of tissue, salivary and serum for early detection and evaluation aggressiveness and occurrence of OSCC were summarized. The emphasis is placed on early detection by tissues, salivary and serum of patients with histologically defined OSCC patients. Although lots of researches for searching OSCC protein biomarker have done, few common protein biomarkers have been detected. Low-redundant protein in tissues, salivary and serum from OSCC may more accurately reflected the progression of OSCC, so novel approach for the depth research strategy and the sample choice for proteomics are of importance in OSCC biomarker discovery.
This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 3
Proteomics - Clinical Applications
Key words oral squamous cell carcinoma (OSCC); proteomics; tumor biomarkers Total number of words: 8847
1 Introduction 1.1 Oral squamouse cell carcinoma characteristics Squamous cell carcinoma is the most common histological type of head and neck cancer, accounting for 90% of all head and neck malignancies[1]. Oral squamous cell carcinoma (OSCC) is a significant cancer worldwide, and
ranks as the eighth most common cancer in males with an average global
incidence of 6.3 per 100,000[1]. Although several therapeutic regimens, including surgery, radiotherapy, and chemotherapy, have been integrally applied for three decades, the 5-year survival rate has only been increasing subtly during the past two decades[2]. Moreover, it is more prevalent in developing countries than in developed countries. This figure is largely due to the advanced stages of the disease at diagnosis which, in turn, limited proper treatments. So far, the most important risk factors identified for OSCC are tobacco use and alcohol consumption, which seem to have a synergistic effect. Recently, it has reported that persistent infection with human papillomavirus (HPV) is also a major risk factor for the development of OSCC, in particular for oropharyngeal squamous cell carcinoma (OPSCC) including tonsillar cancers and base of tongue cancer[3-5]. OSCC follows a similar pattern in its development, and is preceded by precancerous lesions. The malignant potential of oral leukoplakia (OLK), which is the most common type of precancerous type, This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 4
Proteomics - Clinical Applications
was evidenced by the progression from low grade dysplasia, high grade dysplasia, and ultimately to invasive carcinoma. The prognosis for patients with OSCC is largely determined by the stage at presentation and the extent of the tumor, as well as the presence of lymph-node metastases and distant metastases.. Staging of OSCC is determined by clinical examination, imaging, cytology of lymph nodes and definite histopathology after surgery[6]. Disappointingly, survival has not markedly improved in recent decades because patients still frequently develop local-regional recurrences, distant metastases and second primary tumors. The limitedly available information on the molecular carcinogenesis of OSCC or the genetic and biological heterogeneity of the disease has hampered the diagnosis and therapeutic strategies for the development of OSCC. 1.2 The role of biomarker in diagnosis and prognosis of OSCC Patients with early stage OSCC (stage I or II) and non-nodal metastases, who have been treated by surgery or radiotherapy, have excellent 5-year survival rate.However, two thirds of all OSCC patients present with the advanced stage (stage III or IV), due to the fact that large majority of OSCCs arise de novo, without preceding visible changes in the mucosa. Additionally, OSCC may progress from early to advanced stages within a very short time perhaps a few weeks, which hamper early diagnosis for OSCC. During the last decades, progress has been made in the efforts for searching novel treatment modalities for OSCC[7, 8]. Despite these developments also resulting in improved local-regional control and reduced treatment-related morbidity, 5-year survival rate has not improved significantly[9]. Because early detection represents one of the most promising approaches to reducing the growing cancer burden[10], so early diagnosis of primary tumors remains the key to improve treatment outcome. Therefore, deeper
This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 5
Proteomics - Clinical Applications
understanding of the molecular biology of OSCC can provide new insights into its mechanisms of development and progression and it also can provides various biomarkers with a potential application for cancer screening. Alterations in gene sequences, expression levels and protein structure or function have been associated with every type of cancer. These ‘molecular markers’ can be useful in detecting cancer, determining prognosis and monitoring disease progression or therapeutic response[11]. Biomarkers not only can be a single molecular but also a series of molecular，which can be found in a variety of fluids, tissues and cell lines. Biomarkers can be DNA, mRNA, proteins, metabolites, or processes such as apoptosis, angiogenesis or proliferation[12]. The cancer biomarker, which is produced either by the tumor itself or by other tissues, is a molecular signature indicating the physiologic and pathologic changes in a particular tissue or cell type during the development of cancer. There are several types of biomarkers, including diagnostic biomarker, prognostic biomarker and stratification biomarker, which is also be regarded as predictive biomarker. The reported first cancer marker was the light chain of immunoglobulin in the urine, as identified in 75% of patients with myeloma. For example, CA-125 which has been proved to be an prognostic biomarker for ovarian epithelial cancer[14], carcinoembryonic antigen (CEA) which has been proved to be the prognosis biomarker for colon cancer and prostate-specific antigen (PSA) which has been considered to be one of the best prostate cancer markers[15, 16]. Genomic and proteomic technologies have greatly increased the biomarker number and it has been realized that multi-parametric analysis of genes or proteins is especially important for searching biomarkers. mRNA information could not best reflect the function of proteins, which are the functional components, the use of proteomic patterns to enable tumor diagnosis seems to be much more promising.
This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 6
Proteomics - Clinical Applications
Proteins produced by cancer cells or their microenvironment may eventually enter the circulation system and the expression patterns of these proteins could be assessed by mass spectrometry. Proteomics can help us to deeply understand the changes in the levels of multiple proteins involved in oncogenesis and cancer progression and identify new diagnostic or prognostic biomarkers [17]. Mass-spectrometry-based methods of proteomic analysis have improved and include more-advanced technology that brings higher mass accuracy, higher detection capability, and shorter cycling times, thereby enabling increased throughput and more-reliable data[18]. Technologies such as differential in-gel electrophoresis, two-dimensional polyacrylamide gel electrophoresis and multidimensional protein-identification technology can be used for high-throughput protein profiling. The technology that has received considerable attention involves the use of a minute amount of unfractionated serum sample added to a 'protein-chip', which is subsequently analyzed
by
surface-enhanced
laser-desorption–ionization
time-of-flight
mass
spectrometry
(SELDI-TOF-MS) to generate a proteomic signature of serum[19]. Quantitative proteomics is an important branch of proteomics that is applied to quantify and identify all the proteins expressed by a whole genome or in a complex mixture. Comparative proteomics, a study of profiling the global protein expressions within a cell, tissue or organism in comparison to its control, has become one of the promising approaches for the discovery of candidate disease biomarkers.
2 OSCC biomarkers and proteomics The identification of molecular mechanisms underlying cancer initiation and progression could aid in the development of new diagnostic and treatment options for the disease. The processes of cancer cell transformation and progression is extremely complex events involving the deregulation of a variety of genes which controlled cell proliferation, differentiation and cell death. Different proteins, probably in the This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 7
hundreds or thousands, may be up-regulated or down-regulated
Proteomics - Clinical Applications
simultaneously and may conduct
distinct cell functions. Therefore, the analysis of hundreds of proteins simultaneously holds great promise for accurately predicting the function of marker proteins. 2.1. Tissue proteomics analysis for OSCC All the researches on detecting OSCC biomarkers from OSCC tissues by proteomics had been summarized in Table 1. 2.1.1 Protein biomarkers for early detection of OSCC There are fewer researches about discrimination the protein expression profiles between tumor tissue and precancerous lesion with different stages of dysplasia. In the process of disease progress from OLK to OSCC, some underlying molecular signaling network must have been changed. Therefore, in order to identify novel diagnostic targets underlying the malignant transformation from precancerous to cancerous tissue, Chen et al. evaluated protein expression differences by proteomics methods. Six proteins obtained from tumors and OLK tissues with dysplasia were analyzed by two-dimensional gel electrophoresis, followed by ESI-Q-TOF tandem mass spectrometry, then Gene Ontology (GO) analysis and biological networks were applied to identify new therapeutic targets or protein markers. Results identified approximately 85 differentially and constantly expressed proteins, including 52 up-regulated and 33 down-regulated. Three proteosome activators PA28a, b and g were shown to have up-regulated in OSCC cells relative to oral keratinocytes indicating three homologs of PA28 may play an important role in malignant transformation[20]. In order to identify the proteins that are differentially expressed in OSCC compared to adjacent non-tumor tissue, Pitak et al. analyzed fresh tissue samples of cancer and their adjacent normal mucosa from 10 patients with well differentiated OSCC. Five cases were stage I and II (early-stage group) and five This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
cases
were
stage
III
and
IV
Page 8
(late-stage
group).
Proteomics - Clinical Applications
Two-dimensional
(2D)
sodium
dodecyl
sulphate–polyacrylamide gel electrophoresis accompanied by mass spectrometry (matrix-assisted laser desorption/ioni-sation-time-of-flight mass spectrometry and liquid chromatography–tandem mass spectrometry) was used to analyze and identify the differentially expressed proteins. A total of 68 proteins (63 up-regulated, five down-regulated) were differentially expressed in early-stage disease, and 39 proteins (37 up-regulated, two down-regulated) were significantly altered in late-stage disease. Among these, 14 proteins were altered in both groups. A total of 44 proteins were identified, including heat shock proteins (HSPs: Hsp90, HSPA5 and HSPA8), keratins (K1, K6A and K17), tubulin, cofilin 1, 14-3-3s and metabolic enzymes. These proteins are involved in various cellular processes essential for cell growth, survival and cell migration[21]. In order to unveil the molecular signals triggering the onset of oral carcinogens, two-dimensional gel electrophoresis method was used to analyze 10 pairs of tissues T3 (9 N0, 1 N2) including primary OSCC and their matched adjacent normal surrounding mucosa specimens. 41 proteins were identified as commonly over-expressed in OSCC tissues. In OSCC tissues, alphaB-crystallin, tropomyosin 2, myosin light chain 1, HSP27, stratifin, thioredoxin-dependent peroxide reductase, flavin reductase, vimentin, rho GDP-dissociation inhibitor 2 (rho GDI-2), glutathione S-transferase Pi (GST-pi) and superoxide dismutase [Mn] (MnSOD) were significantly over-expressed (an average of 7.2, 6.0, 5.7, 4.3, 3.6, 3.4, 3.0, 3.0, 2.6, 2.5, 2.1-fold, respectively)[22].
Tumor tissues are the reliable sources for searching tumor biomarker. Formalin fixation and tissue embedding in paraffin wax (FFPE) is a universal approach for tissue processing, histologic evaluation, and routine diagnosis, as it preserves the cellular morphology and tissue architecture. Combination of
This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 9
Proteomics - Clinical Applications
proteomic analysis with laser capture microdissection may afford performing the proteomic characterization of normal and pathologic cell populations from clinical specimens, thus reflecting their protein make up as they exist in vivo. Therefore, researchers combined the use of laser capture micro-dissection methods enabling the extraction of peptides directly from FFPE samples, and optimized chromatographic approaches to undertake a large-scale proteomic study to identify proteins expressed in FFPE head and neck squmouse cell carcinoma (HNSCC) tissues. Appropriate paraffin blocks of formalin-fixed tissues from 4 normal squamous epitheliums, and 4 well differentiated (WD), 4 moderately differentiated (MD), and 4 poorly differentiated (PD) HNSCC were retrieved to help identify novel molecular targets for the early detection, prevention, and treatment of HNSCC. A large number of proteins expressed in normal oral epithelium and HNSCC, including cytokeratins, intermediate filaments, differentiation markers, and proteins involved in stem cell maintenance, signal transduction, migration, cell cycle regulation, growth and angiogenesis, matrix degradation, and proteins with tumor suppressive and oncogenic potential, were readily detected. To enable the comparison of protein expression across all tissue samples, they used a spectral count method in which each protein that was identified in any one set was evaluated based on the number of unique peptides identified, and the total number of times that those peptides were observed in five successive runs for each set. The number of proteins identified for each set as a group (all four sample results pooled together) ranged from 391 in the normal oral epithelia to 866 total proteins in the WD HNSCC tumors. As indicated, 115 proteins were identified as shared across all normal and tumor tissues. Cytokeratins and desmosomal proteins, stood out as the most abundant in all four-tissue sets. Most keratins were nearly equally represented across all tissue samples, for example, cytokeratin 5, Cytokeratin 14, cytokeratin 4, desmoplakin, plakophilin 1, periplakin, and desmoglein
This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 10
Proteomics - Clinical Applications
precursors, were prominent in all samples. Also notable the family of calcium-binding proteins (calgranulin A, calgranulin B, S10 0 A14, calmod ulinlike protein), keratinocyte differentiation markers (involucrin, small pro-line-rich protein 3, profilin 1, cornifin A, cornifin B), and many membrane-related molecules (annexin A1, actin-like protein 2) were identified. A number of proteins including heat shock protein 27 (HSP27) (15), HSP70 (16), and glutathioneS -transferase (15), expected to be of higher abundance in tumor samples, were identified. A number of signaling molecules (e.g., Ras GTPase-activating-like protein IQGAP1, obscurin, tyrosine-protein kinase ITK) were also identified. Conversely, the DNA excision repair protein ERCC-5 was found to be less abundant in tumor samples. These data indicated that the analysis of FFPE tissues enables the identification of a broad range of functionally diverse proteins in normal and tumor squamous epithelia, albeit many exhibit a distinct expression profile. Forty-two proteins unique to HNSCC were readily detected by multiple peptides (>10). Among them, the most abundant was vimentin, a protein involved in epithelial-mesenchymal transition. A variety of proteins involved in cell migration, signaling, and proteolysis were also identified. Eighty-five less abundant proteins (i.e., identified by >4 peptides but
